CG000168 Chromium Single Cell ATAC Reagent Kits User Guide Rev A
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CG000168 Rev A USER GUIDE Chromium Single Cell ATAC Reagent Kits FOR USE WITH Chromium Single Cell ATAC Library & Gel Bead Kit, 16 rxns PN-1000110 Chromium Single Cell ATAC Library & Gel Bead Kit, 4 rxns PN-1000111 Chromium Chip E Single Cell ATAC Kit, 48 rxns PN-1000082 Chromium Chip E Single Cell ATAC Kit, 16 rxns PN-1000086 Chromium i7 Multiplex Kit N, Set A, 96 rxns PN-1000084 10xGenomics.com Notices Notices Document Number CG000168 | Rev A Legal Notices © 2018 10X Genomics, Inc. (10x Genomics). All rights reserved. Duplication and/or reproduction of all or any portion of this document without the express written consent of 10x Genomics, is strictly forbidden. Nothing contained herein shall constitute any warranty, express or implied, as to the performance of any products described herein. Any and all warranties applicable to any products are set forth in the applicable terms and conditions of sale accompanying the purchase of such product. 10x Genomics provides no warranty and hereby disclaims any and all warranties as to the use of any third-party products or protocols described herein. The use of products described herein is subject to certain restrictions as set forth in the applicable terms and conditions of sale accompanying the purchase of such product. A non-exhaustive list of 10x Genomics’ marks, many of which are registered in the United States and other countries can be viewed at: www.10xgenomics.com/trademarks. 10x Genomics may refer to the products or services offered by other companies by their brand name or company name solely for clarity, and does not claim any rights in those third-party marks or names. 10x Genomics products may be covered by one or more of the patents as indicated at: www.10xgenomics.com/patents. The use of products described herein is subject to 10x Genomics Terms and Conditions of Sale, available at www.10xgenomics.com/legal-notices, or such other terms that have been agreed to in writing between 10x Genomics and user. All products and services described herein are intended FOR RESEARCH USE ONLY and NOT FOR USE IN DIAGNOSTIC PROCEDURES. Notice to Customer: The listed products and their use are the subject of United States Patent Nos. 6,159,736, and 6,294,385, European Patent No. 1115856 and related patents and patent applications licensed from the Wisconsin Alumni Research Foundation. Instrument & Licensed Software Updates Warranties Updates to existing Instruments and Licensed Software may be required to enable customers to use new or existing products. In the event of an Instrument failure resulting from an update, such failed Instrument will be replaced or repaired in accordance with the 10x Limited Warranty, Assurance Plan or service agreement, only if such Instrument is covered by any of the foregoing at the time of such failure. Instruments not covered under a current 10x Limited Warranty, Assurance Plan or service agreement will not be replaced or repaired. Support Email: support@10xgenomics.com 10x Genomics 7068 Koll Center Parkway Suite 401 Pleasanton, CA 94566 USA Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 2 TOC Table of Contents Introduction 4 Chromium Single Cell ATAC Reagent Kits Chromium Accessories Recommended Thermal Cyclers Additional Kits, Reagents & Equipment Protocol Steps & Timing Stepwise Objectives Tips & Best Practices Step 1 Transposition Nuclei Concentration Guidelines 1.1 Prepare Transposition Mix 1.2 Isothermal Incubation Step 2 GEM Generation & Barcoding 2.1 Prepare Master Mix 2.2 Load Chromium Chip E 2.3 Run the Chromium Controller 2.4 Transfer GEMs 2.5 GEM Incubation Step 3 Post GEM Incubation Cleanup & QC 3.1 Post GEM Incubation Cleanup – Dynabeads 3.2 Post GEM Incubation Cleanup – SPRIselect Step 4 Library Construction 4.1 Sample Index PCR 4.2 Post Sample Index Double Sided Size Selection – SPRIselect 4.3 Post Library Construction QC 4.4 Post Library Construction Quantification Sequencing Troubleshooting 6.1 GEMs 6.2 Chromium Controller Errors Appendix 5 8 8 9 10 11 13 18 19 20 21 21 22 23 24 25 26 26 27 28 29 30 32 33 34 35 36 37 38 39 42 43 45 46 Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 3 Introduction Chromium Single Cell ATAC Reagent Kits Chromium Accessories Additional Kits, Reagents & Equipment Protocol Steps & Timing Stepwise Objectives Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 4 Introduction Chromium Single Cell ATAC Reagent Kits Chromium Single Cell ATAC Library & Gel Bead Kit, 16 rxns PN-1000110 Chromium Single Cell ATAC Library Kit, 16 rxns PN-1000083 (store at −20°C) Chromium Single Cell ATAC Library Kit # PN ATAC Buffer 1 2000122 ATAC Enzyme 1 2000123 Nuclei Buffer 1 2000153 Barcoding Reagent 1 2000124 Barcoding Enzyme 1 2000125 SI-PCR Primer B 1 2000128 Reducing Agent B 1 2000087 Amp Mix 1 2000047 Cleanup Buffer 2 2000088 10xGenomics.com Chromium Single Cell ATAC Gel Bead Kit, 16 rxns PN-1000081 (store at −80°C) Chromium Single Cell ATAC Gel Beads # Single Cell ATAC Gel Beads PN 2 2000132 10xGenomics.com Dynabeads™ MyOne™ SILANE, PN-2000048 (store at 4°C) # Dynabeads MyOne SILANE Click to TOC PN 1 2000048 Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 5 Introduction Chromium Single Cell ATAC Reagent Kits Chromium Single Cell ATAC Library & Gel Bead Kit, 4 rxns PN-1000111 Chromium Single Cell ATAC Library Kit, 4 rxns PN-1000087 (store at −20°C) Chromium Single Cell ATAC Library Kit # PN ATAC Buffer 1 2000122 ATAC Enzyme 1 2000138 Nuclei Buffer 1 2000153 Barcoding Reagent 1 2000124 Barcoding Enzyme 1 2000139 SI-PCR Primer B 1 2000128 Reducing Agent B 1 2000087 Amp Mix 1 2000103 Cleanup Buffer 1 2000088 10xGenomics.com Chromium Single Cell ATAC Gel Bead Kit, 4 rxns PN-1000085 (store at −80°C) Chromium Single Cell ATAC Gel Beads # Single Cell ATAC Gel Beads (4 rxns) PN 1 2000132 10xGenomics.com Dynabeads™ MyOne™ SILANE, PN-2000048 (store at 4°C) # Dynabeads MyOne SILANE Click to TOC PN 1 2000048 Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 6 Introduction Chromium Chip E Single Cell ATAC Kit, 48 rxns PN-1000082 (store at ambient temperature) Chromium Partitioning Oil Partitioning Oil # PN Chromium Recovery Agent 6 220088 Recovery Agent # PN 6 220016 Chromium Chip E & Gaskets # PN Chip E Single Cell ATAC 6 2000121 Gasket, 6-pack 1 370017 10xGenomics.com Chromium Chip E Single Cell ATAC Kit, 16 rxns PN-1000086 (store at ambient temperature) Chromium Partitioning Oil Partitioning Oil # PN Chromium Recovery Agent 2 220088 Recovery Agent # PN 2 220016 Chromium Chip E & Gaskets # PN Chip E Single Cell ATAC 2 2000121 Gasket, 2-pack 1 3000072 10xGenomics.com Chromium i7 Multiplex Kit N, Set A, 96 rxns PN-1000084 (store at −20°C) Chromium i7 Multiplex Kit N Set A Chromium i7 Sample Index Plate N, Set A Click to TOC # PN 1 3000262 Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 7 Introduction Chromium Accessories Recommended Thermal Cyclers Click to TOC Product PN (Orderable) PN (Item) 10x Vortex Adapter 120251 330002 10x Chip Holder 120252 330019 10x Magnetic Separator 120250 230003 Thermal cyclers used must support uniform heating of 100 µl emulsion volumes. Supplier Description Part Number BioRad C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module 1851197 Eppendorf MasterCycler Pro North America 950030010 International 6321 000.01 Thermo Fisher Scientific Veriti 96-Well Thermal Cycler 4375786 Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 8 Introduction Additional Kits, Reagents & Equipment Supplier The items in the table below have been validated by 10x Genomics and are highly recommended for the Chromium Single Cell ATAC protocol. Substituting materials may adversely affect system performance. Description Part Number (US) Plastics Choose either Eppendorf or USA Scientific PCR 8-tube strips. Eppendorf PCR Tubes 0.2 ml 8-tube strips DNA LoBind Tubes, 1.5 ml DNA LoBind Tubes, 2.0 ml 951010022 022431021 022431048 USA Scientific TempAssure PCR 8-tube strip Rainin Tips LTS W-O 200UL Filter RT-L200WFLR Tips LTS 20UL Filter RT-L10FLR Tips LTS 200UL Filter RT-L200FLR Tips LTS 1ML Filter RT-L1000FLR 30389241 30389226 30389240 30389213 Thermo Fisher Scientific Nuclease-free Water AM9937 Corning Cellgro Phosphate-Buffered Saline (PBS) 1X without calcium and magnesium 21-040-CV Millipore Sigma Ethanol, Pure (200 Proof, anhydrous) E7023-500ML Beckman Coulter SPRIselect Reagent Kit B23317 Bio-Rad 10% Tween 20 1662404 Ricca Chemical Company Glycerin (glycerol), 50% (v/v) Aqueous Solution 3290-32 Qiagen Qiagen Buffer EB 19086 Rainin Pipet-Lite Multi Pipette L8-50XLS Pipet-Lite Multi Pipette L8-200XLS 17013804 17013805 VWR Vortex Mixer Divided Polystyrene Reservoirs 10153-838 41428-958 Eppendorf ThermoMixer C 5382000015 1402-4700 Kits & Reagents Equipment Quantification & Quality Control Agilent 2100 Bioanalyzer Laptop Bundle High Sensitivity DNA Kit 4200 TapeStation High Sensitivity D1000 ScreenTape High Sensitivity D1000 Reagents KAPA Biosystems Click to TOC Choose Bioanalyzer or TapeStation, based on availability and preferences. KAPA Library Quantification Kit for Illumina® Platforms Chromium Single Cell ATAC Reagent Kits User Guide | Rev A G2943CA 5067-4626 G2991AA 5067-5592 5067-5593 KK4824 9 Introduction Protocol Steps & Timing Steps Timing Stop & Store Nuclei Isolation 3h 2h Dependent on Cell Type ~1-2 h Step 1 – Transposition 1.1 1.2 Prepare Transposition Mix Isothermal Incubation 10 min 60 min Step 2 – GEM Generation & Barcoding 2.1 2.2 2.3 2.4 2.5 4h Prepare Master Mix Load Chromium Chip E Run the Chromium Controller Transfer GEMs GEM Incubation 10 min 10 min 7 min 3 min 45 min Step 3 – Post GEM Incubation Cleanup & QC 3.1 3.2 Post GEM Incubation Cleanup – Dynabeads Post GEM Incubation Cleanup – SPRIselect 35 min 15 min STOP 4°C ≤2 h or −20°C ≤ 2 weeks STOP 4°C ≤72 h or −20°C long-term Step 4 – Library Construction 6h 4.1 4.2 4.3 Click to TOC Sample Index PCR Post Sample Index Double Sided Size Selection – SPRIselect Post Library Construction QC 45 min 20 min 60 min Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 10 Introduction Stepwise Objectives The Chromium Single Cell ATAC Solution provides a comprehensive, scalable approach to determine the regulatory landscape of chromatin in hundreds to thousands of cells in a single sample. This is achieved by transposing nuclei in a bulk solution; then using a microfluidic chip, the nuclei are partitioned into nanoliter-scale Gel Beads-in-emulsion (GEMs). GemCode Technology samples a pool of ~750,000 10x Barcodes to separately and uniquely index the transposed DNA of each individual cell. Libraries are generated and sequenced, and 10x Barcodes are used to associate individual reads back to the individual partitions, and thereby, to each individual cell. Step 1 Transposition Nuclei suspensions are incubated in a Transposition Mix that includes a Transposase. The Transposase enters the nuclei and preferentially fragments the DNA in open regions of the chromatin. Simultaneously, adapter sequences are added to the ends of the DNA fragments. Step 2 GEM Generation & Barcoding GEMs are generated by combining barcoded Gel Beads, transposed nuclei, a Master Mix, and Partitioning Oil on a Chromium Chip E. To achieve single nuclei resolution, the nuclei are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contains no nuclei, while the remainder largely contain a single nucleus. Gel Beads 10x Barcode P5 Chromium Chip E Gel Beads GEMs 10x Barcoded Gel Beads Upon GEM generation, the Gel Bead is dissolved. Oligonucleotides containing (i) an Illumina® P5 sequence, (ii) a 16 nt 10x Barcode and (iii) a Read 1 (Read 1N) sequence are released and mixed with DNA fragments and Master Mix. Thermal cycling of the GEMs produces 10x barcoded singlestranded DNA. After incubation, the GEMs are broken and pooled fractions are recovered. Nuclei, Enzyme Oil Inside Individual GEMs P5 Read 1N Read 2N 10x Barcode Denaturation, Linear Amplification Linear Amplification Product P5 Click to TOC Read 1N Insert Read 2N 10x Read 1N Barcode 10x Barcoded DNA Fragments Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 11 Introduction Step 3 Post GEM Incubation Cleanup & QC Silane magnetic beads are used to remove leftover biochemical reagents from the post GEM reaction mixture. Solid Phase Reversible Immobilization (SPRI) beads are used to eliminate unused barcodes from the sample. Step 4 Library Construction P7, a sample index, and Read 2 (Read 2N) sequence are added during library construction via PCR. The final libraries contain the P5 and P7 primers used in Illumina® bridge amplification. Pooled Amplified DNA Processed in Bulk P5 Priming P5 Sample Index PCR 10x Read 1N Barcode Sample Index N Insert P7 Read 2N The Chromium Single Cell ATAC protocol produces Illumina® -ready sequencing libraries. Illumina® sequencer compatibility, sample indices, sequencing depth & run parameters, library loading and pooling are summarized. Chromium Single Cell ATAC Library i7:8 Sample Index N i5:16 Read 1N P5 10x Barcode Read 1N Insert Read 2N Read 2N P7 See Appendix for Oligonucleotide Sequences Click to TOC P7 Read 2N P5 Step 5 Sequencing Sample Index N 10x Barcode Read 1N Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 12 Tips & Best Practices TIPS Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 13 Tips & Best Practices Icons TIPS Tips & Best Practices section includes additional guidance Emulsion-safe Plastics Multiplet Rate General Reagent Handling ! Signifies critical step requiring accurate execution Troubleshooting section includes additional guidance • Use 10x Genomics validated emulsion-safe plastic consumables when handling GEMs as some plastics can destabilize GEMs. Multiplet Rate (%) # of Nuclei Loaded # of Nuclei Recovered 0.4% ~775 ~500 0.8% ~1,550 ~1,000 1.6% ~3,075 ~2,000 2.3% ~4,625 ~3,000 3.1% ~6,150 ~4,000 3.9% ~7,700 ~5,000 4.6% ~9,250 ~6,000 5.4% ~10,750 ~7,000 6.2% ~12,300 ~8,000 6.9% ~13,850 ~9,000 7.7% ~15,400 ~10,000 • Fully thaw and thoroughly mix reagents before use. • Keep all enzymes and Master Mixes on ice during setup and use. Promptly move reagents back to the recommended storage. • Calculate reagent volumes with 10% excess of 1 reaction values. • Cover Partitioning Oil tubes and reservoirs to minimize evaporation. • Thoroughly mix samples with the beads during bead-based cleanup steps. 50% Glycerol Solution • Purchase 50% glycerol solution from Ricca Chemical Company, Glycerin (glycerol), 50% (v/v) Aqueous Solution, PN-3290-32. • Prepare 50% glycerol solution: i. Mix an equal volume of water and 99% Glycerol, Molecular Biology Grade. ii. Filter through a 0.2-μm filter. iii. Store at −20°C in 1-ml LoBind tubes. 50% glycerol solution should be equilibrated to room temperature before use. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 14 Tips & Best Practices Pipette Calibration • Follow manufacturer’s calibration and maintenance schedules. Chromium Chip Handling • Minimize exposure of reagents, chips, and gaskets to sources of particles and fibers, laboratory wipes, frequently opened flip-cap tubes, clothing that sheds fibers, and dusty surfaces. • Pipette accuracy is particularly important when using SPRIselect reagents. • Execute steps without pause or delay, unless indicated. When multiple chips are to be used, load, run, and collect the content from one chip before loading the next. • Fill all unused input wells in rows labeled 1, 2, and 3 on a chip with an appropriate volume of 50% glycerol solution before loading the used wells. DO NOT add glycerol to the Recovery Wells. • Avoid contacting the bottom surface of the chip with gloved hands and other surfaces. Frictional charging can lead to inadequate priming of the channels, potentially leading to either clogs or wetting failures. • Minimize the distance that a loaded chip is moved to reach the Chromium Controller. • Keep the chip horizontal to prevent wetting the gasket with oil, which depletes the input volume and may adversely affect the quality of the assay. 10x Chip Holders • 10x Chip Holders encase Chromium Chips. • The holder lid flips over to become a stand, holding the chip at 45 degrees for optimal Recovery Well content removal. • Squeeze the black sliders on the back side of the holder together to unlock the lid and return the holder to a flat position. Chromium Chip & Holder Assembly 10X Chip Holder Clip Guide Sliders • Align notch on the chip (upper left corner) and the holder. • Insert the left-hand side of the chip under the guide. Depress the right-hand side of the chip until the spring-loaded clip engages. 10X Chip Holder Chromium Chip Assembled Chip • Close the lid before dispensing reagents into the wells. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 15 Tips & Best Practices Chromium Chip Loading • Place the assembled chip and holder flat on the bench with the lid closed. • Dispense at the bottom of the wells without introducing bubbles. • Wait for the Cell Bead Mix to drain into the bottom of the pipette tips and dispense again to ensure complete volume transfer. • Refer to Load Chromium Chip E for specific instructions. Gel Bead Handling • Use one tube of Gel Beads per sample. DO NOT puncture the foil seals of tubes not used at the time. • Equilibrate the Gel Beads strip to room temperature before use. • Store unused Gel Beads at −80°C and avoid more than 12 freeze-thaw cycles. DO NOT store Gel Beads at −20°C. • Attach a 10x Vortex Adapter to the top of standard laboratory vortexers to vortex the Gel Bead strips. • After vortexing, remove the Gel Bead strip from the adapter. Flick the Gel Bead strip in a sharp, downward motion to maximize Gel Bead recovery. Confirm there are no bubbles at the bottom of the tubes. • If the required volume of beads cannot be recovered, place the pipette tips against the sidewalls and slowly dispense the Gel Beads back into the tubes. DO NOT introduce bubbles into the tubes and verify that the pipette tips contain no leftover Gel Beads. Withdraw the full volume of beads again by pipetting slowly. 10x Gasket Attachment • After reagents are loaded, attach the gasket by holding the tongue (curved end, to the right) and hook it on the left-hand tabs of the holder. Gently pull the gasket toward the right and hook it on the two right-hand tabs. Notched Cut • DO NOT touch the smooth side of the gasket. DO NOT press down on the top of the gasket after attachment. Tongue • Keep the assembly horizontal to avoid wetting the gasket with Partitioning Oil. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 16 Tips & Best Practices 10x Magnetic Separator SPRIselect Cleanup & Size Selection • Offers two positions of the magnets (high and low) relative to a tube, depending on its orientation. Flip the magnetic separator over to switch between high (magnet•High) or low (magnet•Low) positions. • After aspirating the desired volume of SPRIselect reagent, examine the pipette tips before dispensing to ensure the correct volume is transferred. • Pipette mix thoroughly as insufficient mixing of sample and SPRIselect reagent will lead to inconsistent results. • Use fresh preparations of 80% Ethanol. Tutorial — SPRIselect Reagent:DNA Sample Ratios SPRI beads selectively bind DNA according to the ratio of SPRIselect reagent (beads). Example: Ratio = Volume of SPRIselect reagent added to the sample = 50 µl = 0.5X Volume of DNA sample 100 µl Schematic of Double Sided Size Selection After the first SPRI, supernatant is transferred for a second SPRI while larger fragments are discarded (green). After the second SPRI, fragments on beads are eluted and kept while smaller fragments are discarded (blue). Final sample has a tight fragment size distribution with reduced overall amount (black). Tutorial — Double Sided Size Selection Step a – First SPRIselect: Add 50 µl SPRIselect reagent to 100 µl sample (0.5X). Ratio = Volume of SPRIselect reagent added to the sample = 50 µl = 0.5X Volume of DNA sample 100 µl Step b – Second SPRIselect: Add 30 µl SPRIselect reagent to supernatant from step a (0.8X). Ratio = Total Volume of SPRIselect reagent added to the sample (step a + b) = 50 µl + 30 µl = 0.8X Sample Indices in Sample Index PCR Original Volume of DNA sample 100 µl • Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. • Each well in the i7 Sample Index plate N contains a unique mix of 4 oligos. • The sample indexes can therefore be used in any combination. • Each sample index set is base-balanced to avoid monochromatic signal issues when it is the sole sample loaded on an Illumina® sequencer. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 17 Step 1 Transposition 1.1 1.2 Prepare Transposition Mix Isothermal Incubation 1 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 18 Step 1 1.0 Transposition Transposition GET STARTED! Action Equilibrate to Room Temperature Item 10x PN Preparation & Handling ATAC Buffer 2000122 Vortex, centrifuge briefly. −20°C Nuclei Buffer* 2000153 Thaw. Vortex, centrifuge briefly. −20°C 2000123/ 2000138 Centrifuge briefly. −20°C *Concentrated 20X stock; dilute 1:20 in nuclease-free water before use. (See below to Prepare Diluted Nuclei Buffer) Place on Ice ATAC Enzyme Storage Nuclei** in Diluted Nuclei Buffer (See below to Prepare Diluted Nuclei Buffer) ! ! Prepare **Refer to Demonstrated Protocol Nuclei Isolation for ATAC Sequencing (Document CG000167) for isolating nuclei. Adhering to this protocol is critical for optimal assay performance. If following a different nuclei isolation protocol, use the Diluted Nuclei Buffer for final nuclei pellet suspension. The use of the Tris-based Diluted Nuclei Buffer for nuclei suspension is critical for optimal assay performance. The composition of the Diluted Nuclei Buffer, including Magnesium concentration, has been optimized for the Transposition and Barcoding steps. Suspension of nuclei in a different buffer may not be compatible with the downstream protocol steps. Diluted Nuclei Buffer Diluted Nuclei Buffer Nuclei Buffer (PN-2000153) Nuclease-free Water Click to TOC Stock Final 1 ml 20X 1X 50 µl - - 950 µl Maintain at 4°C Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 19 Step 1 Nuclei Concentration Guidelines Transposition Based on the Targeted Nuclei Recovery, resuspend the nuclei in Diluted Nuclei Buffer to get corresponding Nuclei Stock Concentrations (see Table). This enables pipetting volumes of the Nuclei Stock for Transposition (step 1.1) to be 2-5 µl. Higher Nuclei Stock Concentrations will result in lower pipetting volumes that may increase nuclei input variability. Targeted Nuclei Recovery Nuclei Stock Concentration (nuclei/µl) 500 155-390 1,000 310-780 2,000 610-1,540 3,000 925-2,300 4,000 1,230-3,075 5,000 1,540-3,850 6,000 1,850-4,600 7,000 2,150-5,400 8,000 2,460-6,150 9,000 2,770-6,900 10,000 3,080-7,700 Calculate volume of Nuclei Stock and Diluted Nuclei Buffer for a total volume of 5 µl Volume of Nuclei Stock (µl) = Targeted Nuclei Recovery x 1.53 (Recovery efficiency factor) Nuclei Stock Concentration (nuclei/ µl) Volume of Diluted Nuclei Buffer* (µl) = 5 µl - volume of Nuclei Stock (µl) *Use ONLY Diluted Nuclei Buffer (Dilute Nuclei Buffer (PN-2000153) 1:20 in nuclease-free water) Example Calculation Targeted Nuclei Recovery = 4000 nuclei Nuclei Stock Concentration = 2500 nuclei/ µl Recovery efficiency factor 1.53 Volume of Nuclei Stock (µl) = Targeted Nuclei Recovery x 1.53 (Recovery efficiency factor) = 4000 x 1.53 = 2.45 µl Nuclei Stock Concentration (nuclei/µl) 2500 Volume of Diluted Nuclei Buffer = 5 µl - 2.45 ul = 2.55 µl Add calculated volumes of Diluted Nuclei Buffer and Nuclei Stock to the Transposition Mix in step 1.1 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 20 Step 1 Transposition 1.1 Prepare Transposition Mix a. Prepare Transposition Mix on ice. Pipette mix 10x and centrifuge briefly. Transposition Mix Add reagents in the order listed PN 1X (μl) 4X + 10% (μl) 8X + 10% (μl) ATAC Buffer 2000122 7.0 30.8 61.6 ATAC Enzyme 2000123/ 2000138 3.0 13.2 26.4 - 10.0 44.0 88.0 Total b. Add 10 µl Transposition Mix to a tube of a PCR 8-tube strip for each sample. Centrifuge briefly and maintain on ice. c. Refer to Nuclei Concentration Guidelines to calculate the volume of Nuclei Stock and Diluted Nuclei Buffer for a total volume of 5 µl. d. Add the calculated volume of Diluted Nuclei Buffer to the Transposition Mix. Pipette mix. Centrifuge briefly. ! 1.2 Isothermal Incubation Click to TOC e. Gently pipette mix the Nuclei Stock. Add the calculated volume of the Nuclei Stock to the tube containing the Transposition Mix. Gently pipette mix 6x (pipette set to 10 µl). DO NOT centrifuge. a. Incubate in a thermal cycler using the following protocol. Lid Temperature Reaction Volume 50°C 15 µl 60 min Step Temperature Time Incubate 37°C 00:60:00 Hold 4°C Hold Chromium Single Cell ATAC Reagent Kits User Guide | Rev A Run Time 21 Step 2 GEM Generation & Barcoding 2.1 2.2 2.3 2.4 2.5 Prepare Reaction Mix Load Chromium Chip E Run the Chromium Controller Transfer GEMs GEM Incubation 2 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 22 Step 2 2.0 GEM Generation & Barcoding GEM Generation & Barcoding GET STARTED! Action Item 10x PN Preparation & Handling Storage Chromium Single Cell ATAC Gel Beads 2000132 Equilibrate to room temperature 30 min before loading the chip. −80°C Nuclease-free Water - - - Reducing Agent B 2000087 Thaw, vortex, verify no precipitate, centrifuge briefly. −20°C Barcoding Reagent 2000124 Thaw, vortex, verify no precipitate, centrifuge briefly. −20°C Place on Ice Barcoding Enzyme 2000125/ 2000139 Maintain on ice. Store at −20°C immediately after use. −20°C Obtain Partitioning Oil 220088 - Ambient Chromium Chip E 2000121 See Tips & Best Practices. Ambient 10x Gasket 370017/ 3000072 See Tips & Best Practices. Ambient 10x Vortex Adapter 330002 See Tips & Best Practices. Ambient 10x Chip Holder 330019 See Tips & Best Practices Ambient 50% glycerol solution - See Tips & Best Practices. - Equilibrate to Room Temperature ! Firmware Version 3.16 or higher is required in the Chromium Controller or the Single Cell Chromium Controller used for the Single Cell ATAC protocol. Click to TOC If using <8 reactions Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 23 GEM Generation & Barcoding Step 2 2.1 Prepare Master Mix a. Prepare Master Mix on ice. Pipette mix 10x and centrifuge briefly. Master Mix Add reagents in the order listed PN 1X (μl) 4X + 10% (μl) 8X + 10% (μl) Barcoding Reagent 2000124 61.5 270.6 541.2 Reducing Agent B 2000087 1.5 6.6 13.2 Barcoding Enzyme 2000125/ 2000139 2.0 8.8 17.6 - 65.0 286.0 572.0 Total Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 24 Step 2 GEM Generation & Barcoding 2.2 Load Chromium Chip E See Tips & Best Practices for chip handling instructions. When loading the chip, raising and depressing the pipette plunger should each take ~5 sec. When dispensing, raise the pipette tips at the same rate as the liquid is rising, keeping the tips slightly submerged. TIPS a. Assemble Chromium Chip E in a 10x Chip Holder See Tips & Best Practices b. Dispense 50% Glycerol Solution into Unused Chip Wells (if <8 samples per chip) i. 75 μl into unused wells in row labeled 1. DO NOT add 50% glycerol solution to the ii. 40 μl into unused wells in row labeled 2. top row of Recovery Wells. DO NOT use any iii. 240 μl into unused wells in row labeled 3. substitute for 50% glycerol solution. c. Prepare Master Mix + Transposed Nuclei Add 65 µl Master Mix to each tube containing Transposed Nuclei for a total of 80 µl in each tube. d. Load Row Labeled 1 Gently pipette mix the Master Mix + Transposed Nuclei. Using the same pipette tip, dispense 75 µl Master Mix + Transposed Nuclei into the bottom center of each well in row labeled 1 without introducing bubbles. Wait 30 sec. 75 µl Master Mix + Transposed Nuclei If volume is <75 μl, load available volume, which should not be <70 μl. e. Prepare Gel Beads Snap the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec. Remove the Gel Bead strip and flick in a sharp, downward motion to ensure maximum recovery. Confirm there are no bubbles at the bottom of the tubes and liquid levels look even. f. Load Gel Beads in Row Labeled 2 Puncture the foil seal of the Gel Bead tubes. Slowly aspirate 40 µl Gel Beads. Dispense into the bottom of each well in row labeled 2 without introducing bubbles. ! g. Load Row Labeled 3 Dispense 240 µl Partitioning Oil into each well in row labeled 3. Failure to add Partitioning Oil can damage the Chromium Controller. h. Attach 10x Gasket Align the notch with the top left-hand corner. Ensure the gasket holes are aligned with the wells. Avoid touching the smooth gasket surface. DO NOT press down on the gasket. Click to TOC 40 µl Gel Beads 240 µl Partitioning Oil Keep horizontal to avoid wetting the gasket. Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 25 GEM Generation & Barcoding Step 2 2.3 Run the Chromium Controller a. Press the eject button on the Controller to eject the tray. b. Place the assembled chip with the gasket in the tray. Press the button to retract the tray. c. Confirm the program on screen. Press the play button. ! 2.4 Transfer GEMs d. At completion of the run (~7 min), the Controller will chime. Immediately proceed to the next step. a. Place a PCR 8-tube strip on ice. Expose Wells at 45 Degrees b. Press the eject button of the Controller to remove the chip. c. Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees. d. Check the volume in rows 1-3. Abnormally high volume in any well indicates a clog. e. Slowly aspirate 100 µl GEMs from the lowest points of the Recovery Wells without creating a seal between the pipette tips and the wells. Transfer GEMs f. Withdraw pipette tips from the wells. GEMs should appear opaque and uniform across all channels. Excess Partitioning Oil (clear) in the pipette tips indicates a potential clog. g. Over the course of ~20 sec, dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the wells. GEMs h. If multiple chips are run back-to-back, cap/ cover the GEM-containing tube strip or plate and place on ice for no more than 1 h. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 26 GEM Generation & Barcoding Step 2 2.5 GEM Incubation Use a thermal cycler that can accommodate at least 100 µl volume. A volume of 125 µl is the preferred setting on Bio-Rad C1000 Touch. In alternate thermal cyclers, use highest reaction volume setting. a. Incubate in a thermal cycler with the following protocol. Click to TOC Lid Temperature Reaction Volume Run Time 105°C 125 µl 30 min Step Temperature Time 1 72°C 00:05:00 2 98°C 00:00:30 3 98°C 00:00:10 4 59°C 00:00:30 5 72°C 00:01:00 Go to step 3, repeat 11X (Total 12 cycles) 6 15°C Hold Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 27 Step 3 Post GEM Incubation Cleanup & QC 3.1 3.2 Post GEM Incubation Cleanup – Dynabeads Post GEM Incubation Cleanup – SPRIselect 3 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 28 Step 3 3.0 Post GEM Incubation Cleanup & QC Post GEM Incubation Cleanup & QC GET STARTED! Action Item 10x PN Preparation & Handling Storage Reducing Agent B 2000087 Thaw, vortex, verify no precipitate, centrifuge briefly. −20°C Nuclease-free Water - - - Dynabeads MyOne SILANE 2000048 Vortex thoroughly (≥30 sec) to resuspend beads immediately before use. 4°C Beckman Coulter SPRIselect Reagent - Manufacturer’s recommendations. - Thaw at 65°C Cleanup Buffer 2000088 Thaw for 10 min at 65°C at max speed on a thermomixer. Verify there are no visible crystals. Cool to room temperature. −20°C Obtain Recovery Agent 220016 - Ambient Qiagen Buffer EB - Manufacturer’s recommendations. - Bio-Rad 10% Tween 20 - Manufacturer’s recommendations. - 10x Magnetic Separator 230003 - Ambient Prepare 80% Ethanol - Prepare fresh. - Equilibrate to Room Temperature Prepare 10 ml for 8 reactions Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 29 Post GEM Incubation Cleanup & QC Step 3 a. Add 125 µl Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex the biphasic mixture. Gently invert tube 10x to mix. Centrifuge briefly. 3.1 Post GEM Incubation Cleanup – Dynabeads Biphasic Mixture The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque). A smaller aqueous phase volume indicates a clog during GEM generation. ! Remove Recovery Agent b. Slowly remove 125 µl Recovery Agent/ Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample. c. Prepare Dynabeads Cleanup Mix. Dynabeads Cleanup Mix Add reagents in the order listed PN 1X (μl) 4X + 10% (μl) 8X + 10% (μl) 2000088 182 800.8 1601.6 Vortex thoroughly (≥30 sec) immediately before adding to the mix. 2000048 8 35.2 70.4 Reducing Agent B 2000087 5 22 44 Nuclease-free Water - 5 22 44 Total - 200 880 1760 Cleanup Buffer Dynabeads MyOne SILANE d. Vortex and add 200 µl to each sample. Pipette mix 5x (pipette set to 200 µl). Add Dynabeads Cleanup Mix e. Incubate 10 min at room temperature. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 30 Post GEM Incubation Cleanup & QC Step 3 f. Prepare Elution Solution I. Vortex and centrifuge briefly. Elution Solution I Add reagents in the order listed PN 1X (μl) 4X + 10% (μl) 8X + 10% (μl) Buffer EB - 98.0 431.2 862.4 10% Tween 20 - 1.0 4.4 8.8 200087 1.0 4.4 8.8 - 100.0 440.0 880.0 Reducing Agent B Total TIPS g. At the end of 10 min incubation, place on the 10x Magnetic Separator, high position (magnet•High) until the solution clears. h. Remove the supernatant. i. Add 300 µl freshly prepared 80% ethanol to the pellet while on the magnet•High. Wait 30 sec. j. Remove the ethanol. k. Add 200 µl 80% ethanol to pellet. Wait 30 sec. l. Remove the ethanol. m. Centrifuge briefly. Place on the magnet•Low. n. Remove remaining ethanol. o. Remove from the magnet. Immediately add 40.5 µl Elution Solution I to avoid clumping. p. Pipette mix (pipette set to 40 µl) without introducing bubbles. q. Incubate 1 min at room temperature. r. Centrifuge briefly. Place on the magnet•Low until the solution clears. s. Transfer 40 µl sample to a new tube strip. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 31 Step 3 Post GEM Incubation Cleanup & QC a. Vortex the SPRIselect reagent until fully resuspended. Add 48 µl SPRIselect reagent to each sample. Pipette mix thoroughly. 3.2 Post GEM Incubation Cleanup – SPRIselect b. Incubate 5 min at room temperature. c. Centrifuge briefly. Place on the magnet•High until the solution clears. d. Remove the supernatant. e. Add 200 µl 80% ethanol to the pellet. Wait 30 sec. f. Remove the ethanol. g. Repeat steps e and f for a total of 2 washes. h. Centrifuge briefly. Place on the magnet•Low. i. Remove any remaining ethanol. j. Remove the tube strip from the magnet. Immediately add 40.5 µl Buffer EB. k. Pipette mix (pipette set to 30 µl) without introducing bubbles. l. Incubate 2 min at room temperature. m.Centrifuge briefly. Place on the magnet•Low until the solution clears. n. Transfer 40 µl sample to a new tube strip. STOP Click to TOC o. Store at 4°C for up to 72 h or at −20°C for up to 2 weeks, or proceed to the next step. Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 32 Step 4 Library Construction 4.1 4.2 4.3 4.4 Sample Index PCR Post Sample Index Double Sided Size Selection – SPRIselect Post Library Construction QC Post Library Construction Quantification 4 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 33 Step 4 4.0 Library Construction Library Construction GET STARTED! Action Equilibrate to Room Temperature Item 10x PN Preparation & Handling Storage Chromium i7 Sample Index Plate N, Set A 3000262 - −20°C Beckman Coulter SPRIselect Reagent - Manufacturer’s recommendations. - Agilent Bioanalyzer DNA kit - Manufacturer’s recommendations. - SI-PCR Primer B 2000128 Vortex, centrifuge briefly. −20°C Amp Mix 2000047/ 2000103 Vortex, centrifuge briefly. −20°C KAPA Library Quantification Kit for Illumina® Platforms - Manufacturer’s recommendations. - Qiagen Buffer EB - - Ambient 10x Magnetic Separator 230003 See Tips & Best Practices. Ambient Prepare 80% Ethanol - Prepare fresh. Ambient (if used for QC) Place on Ice Obtain Prepare 10 ml for 8 reactions Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 34 Step 4 Library Construction 4.1 Sample Index PCR Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. a. Prepare Sample Index PCR Mix. Sample Index PCR Mix Add reagents in the order listed PN 1X (μl) 4X + 10% (μl) 8X + 10% (μl) Amp Mix 2000047/ 2000103 50 220 440 SI- PCR Primer B 2000128 7.5 33 66 - 57.5 253 506 Total b. Add 57.5 µl Sample Index PCR Mix to 40 µl sample. Pipette mix and centrifuge briefly. c. Add 2.5 µl of an individual Chromium i7 Sample Index N, Set A to each well. Record assignment. Pipette mix and centrifuge briefly. d. Incubate in a thermal cycler with the following protocol. Lid Temperature Reaction Volume Run Time 105°C 100 µl ~30 min Step Temperature Time 1 98°C 00:00:45 2 98°C 00:00:20 3 67°C 00:00:30 4 72°C 00:00:20 Go to step 2, see table below for # cycles 5 72°C 00:01:00 6 4°C Hold Cycle Number Optimization Table The table recommends a starting point for cycle number optimization for cell lines and primary cells based on Targeted Nuclei Recovery. STOP Click to TOC Targeted Nuclei Recovery Cell Lines Total Cycles Primary Cells Total Cycles 500-2,000 12 13 2,001-6,000 11 12 6,001-10,000 10 11 e. Store at 4°C for up to 72 h or proceed to the next step. Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 35 Step 4 Library Construction 4.2 TIPS Post Sample Index Double Sided Size Selection – SPRIselect a. Vortex to resuspend SPRIselect reagent. Add 40 μl SPRIselect reagent to each sample. Pipette mix. b. Incubate 5 min at room temperature. c. Place on the magnet•High until the solution clears. d. Transfer 130 µl supernatant to a new strip tube. DO NOT discard the supernatant. e. Vortex to resuspend SPRIselect reagent. Add 74 µl SPRIselect reagent to each sample. Pipette mix. f. Incubate 5 min at room temperature. g. Place on the magnet•High until the solution clears. h. Remove the supernatant. i. Add 200 µl 80% ethanol to the pellet. Wait 30 sec. j. Remove the ethanol. k. Repeat steps i and j for a total of 2 washes. l. Centrifuge briefly. Place on the magnet•Low. m. Remove remaining ethanol. n. Remove from the magnet. Immediately add 20.5 µl Buffer EB. Pipette mix. o. Incubate 2 min at room temperature. p. Centrifuge briefly. Place on the magnet•Low until the solution clears. q. Transfer 20 µl sample to a new tube strip. STOP Click to TOC r. Store at 4°C for up to 72 h or at −20°C for long-term storage. Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 36 Step 4 4.3 Post Library Construction QC Library Construction a. EITHER Run 1 μl sample on the Agilent Bioanalyzer High Sensitivity DNA chip to determine fragment size. Representative Trace b. OR Run 2 μl sample on the Agilent TapeStation D1000 ScreenTape to determine fragment size. Representative Trace Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 37 Step 4 4.4 Post Library Construction Quantification Library Construction a. Thaw KAPA Library Quantification Kit for Illumina® Platforms. b. Dilute 1 µl sample with deionized water to appropriate dilutions that fall within the linear detection range of the KAPA Library Quantification Kit for Illumina® Platforms. (For more accurate quantification, make the dilution(s) in duplicate). c. Make enough Quantification Master Mix for the DNA dilutions per sample and the DNA Standards (plus 10% excess) using the guidance for 1 reaction volume below. Quantification Master Mix 1X (µl) SYBR Fast Master Mix + Primer 12 Water 4 Total 16 d. Dispense 16 μl Quantification Master Mix for sample dilutions and DNA Standards into a 96 well PCR plate. e. Add 4 μl sample dilutions and 4 μl DNA Standards to appropriate wells. Centrifuge briefly. f. Incubate in a thermal cycler with the following protocol. Step Temperature Run Time 1 95°C 00:03:00 2 95°C 00:00:05 3 67°C 00:00:30 4 Go to Step 2, 29X (Total 30 cycles) g. Follow the manufacturer’s recommendations for qPCR-based quantification. For library quantification for sequencer clustering, determine the concentration using the average size in the region of 150-1000 bp. For Library Construction related questions, contact support@10xgenomics.com Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 38 Sequencing 5 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 39 Step 5 Sequencing Libraries Sequencing Chromium Single Cell ATAC libraries comprise double stranded DNA fragments which begin with P5 and end with P7. Sequencing these libraries produces a standard i7:8 Illumina® BCL data output folder. i5:16 bp P5 10x Barcode Sample Index N Read 1N Insert Read 1N Read 2N Read 2N P7 The BCL data for Single Cell ATAC libraries include: • Paired-end Read 1N containing insert sequence only • Read 2N containing insert sequence, starting from the opposite end of fragment • 8 bp sample index in the i7 read • 16 bp 10X barcode sequence in the i5 read The Cell Ranger scATAC pipeline performs demultiplexing and leverages the 10x Barcodes to group read-pairs and associate them to individual cells for secondary analysis and visualization. In addition to performing standard analysis steps such as alignment, Cell Ranger scATAC leverages the 10x Barcodes to generate chromatin accessibility data with single cell resolution. This enables applications including cell clustering, cell type classification, and differential accessibility at a scale of hundreds to thousands of cells. Illumina® Sequencer Compatibility The compatibility of the listed sequencers has been verified by 10x Genomics. Some variation in assay performance is expected based on sequencer choice. For more information about performance variation, visit the 10x Genomics Support website. • • • • • Sample Indices Sequencing Depth & Run Parameters MiSeq™ NextSeq™ 500/550 (High Output) HiSeq 2500™ (Rapid Run) HiSeq™ 3000/4000 NovaSeq™ Each sample index in the Chromium i7 Sample Index Plate Kit N, Set A (PN-3000262) is a mix of 4 different sequences to balance across all 4 nucleotides. If multiple samples are pooled in a sequence lane, the sample index name (i.e. the Chromium i7 Sample Index Plate N, Set A well ID) is needed in the sample sheet used for generating FASTQs with “cellranger-dna mkfastq”. Sequencing Depth (25,000 reads for Read 1N; 25,000 reads for Read 2N) Sequencing Type Paired-end, dual indexing Sequencing Read Recommended Number of Cycles Read 1N i7 Index i5 Index Read 2N Click to TOC 25,000 read pairs per nucleus 50 cycles 8 cycles 16 cycles 50 cycles Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 40 Step 6 Library Loading Sequencing Once quantified and normalized, Single Cell ATAC libraries should be denatured and diluted according to the table below. Consult the Technical Note on Sequencing Metrics and Base Composition of Chromium Single Cell ATAC Libraries (Document CG000181), available at the 10x Genomics Support website, for more information. Instrument Library Pooling Click to TOC Loading Concentration (pM) PhiX (%) MiSeq™ 11 1 NextSeq™ 500 1.7 1 HiSeq™ 2500 (RR) 11 1 HiSeq™ 4000 180 1 NovaSeq™ 250 1 Pooling dissimilar libraries may compromise the ability to pool effectively due to differences in insert sizes. DO NOT pool Single Cell ATAC libraries with other 10x Genomics libraries. Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 41 Troubleshooting 6 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 42 Troubleshooting 6.1 GEMs STEP 2.4 d After Chip E is removed from the Controller and the wells are exposed NORMAL A B C D E F REAGENT CLOGS & WETTING FAILURES G A H All 8 Recovery Wells are similar in volume and opacity. B C D E F G H Recovery Well G indicates a reagent clog. Recovery Well C and E indicate a wetting failure. Recovery Wells B, D, and F are normal. Wells A and H contain 50% Glycerol Solution. The image indicates clogs in the Gel Bead line (orange arrow) and the sample line (yellow arrow) as evidenced by higher than usual volumes in the input wells. 2.4 e Transfer GEMs from Chip E Recovery Wells A All liquid levels are similar in volume and opacity without air trapped in the pipette tips. Click to TOC B C D E F G H Pipette tips C and E indicate a wetting failure. Pipette tip C contains partially emulsified GEMs. Emulsion is absent in pipette tip E. Pipette tip G indicates a reagent clog. Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 43 Troubleshooting STEP NORMAL REAGENT CLOGS & WETTING FAILURES 3.1 a After transfer of the GEMs + Recovery Agent A B C D E F G A H All liquid levels are similar in the aqueous sample volume (clear) and Recovery Agent/Partitioning Oil (pink). B C D E F G H Tube G indicates a reagent clog has occurred. There is a decreased volume of aqueous layer (clear). Tube C and E indicate a wetting failure has occurred. There is an abnormal residual volume of Recovery Agent/Partitioning Oil (pink). 3.1 b After aspiration of Recovery Agent/ Partitioning Oil A B C D E F G H All liquid volumes are similar in the aqueous sample volume (clear) and residual Recovery Agent/Partitioning Oil (pink). A B C D E F G H Tube G indicates a reagent clog has occurred. There is a decreased volume of aqueous layer (clear). There is also a greater residual volume of Recovery Agent/Partitioning Oil (pink). Tube C and E indicate a wetting failure has occurred. There is an abnormal residual volume of Recovery Agent/Partitioning Oil (pink). 3.1 d After addition of Dynabeads Cleanup Mix A B C D E F G H All liquid volumes are similar after addition of the Dynabeads Cleanup Mix. A B C D E F G H Tube G indicates a reagent clog has occurred. There is an abnormal ratio of Dynabeads Cleanup Mix (brown) to Recovery Agent/Partitioning Oil (appears white). Tube C and E indicate a wetting failure has occurred. There is an abnormal ratio of Dynabeads Cleanup Mix (brown) to Recovery Agent/Partitioning Oil (appears white). If a channel clogs or wetting failure occurs during GEM generation, it is recommended that the sample be remade. If any of the listed issues occur, take a picture and send it to support@10xgenomics.com for further assistance. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 44 Troubleshooting 6.2 Chromium Controller Errors If the Chromium Controller or the Chromium Single Cell Controller fails to start, an error tone will sound and one of the following error messages will be displayed: a. Chip not read – Try again: Eject the tray, remove and/or reposition the 10x Chip Holder assembly and try again. If the error message is still received after trying this more than twice, contact support@10xgenomics.com for further assistance. b. Check gasket: Eject the tray by pressing the eject button to check there is a 10x Gasket on the Chromium Chip. In the case when the 10x Gasket installation was forgotten, install and try again. In the case when a 10x Gasket was already installed, remove, reapply, and try again. If the error message is still received after trying either of these more than twice, contact support@10xgenomics.com for further assistance. c. Pressure not at Setpoint: i. If this message is received within a few seconds of starting a run, eject the tray by pressing the eject button and check for dirt or deposits on the 10x Gasket. If dirt is observed, replace with a new 10x Gasket and try again. If the error message is still received after trying this more than twice, contact support@10xgenomics.com for further assistance. ii. If this message is received after a few minutes into the run, the Chromium Chip must be discarded. Do not try running this Chromium Chip again as this may damage the Chromium Controller. d. CAUTION: Chip Holder not Present: Eject the tray by pressing the eject button to check there is a 10x Chip Holder encasing the Chromium Chip. In the case when the 10x Chip Holder was forgotten, install with a 10x Gasket in place, and try again. If the error message is still received after a 10x Chip Holder is confirmed as in place, contact support@10xgenomics.com for further assistance. e. Invalid Chip CRC Value: This indicates the Chromium Chip has encountered an error, should not be run, and must be discarded. Contact support@10xgenomics.com for further assistance. Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 45 Appendix Oligonucleotide Sequences 7 Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 46 Appendix Oligonucleotide Sequences Protocol steps correspond to the Chromium Single Cell ATAC Reagent Kits User Guide (CG000168) Protocol Step 1 – Transposition Transposition Mix Read 1N primer sequence: Read 2N primer sequence: 5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3ʹ 5ʹ-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3ʹ Transposed DNA Product Read 1N Read 2N 5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-----insert-- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3ʹ 3ʹ-AGCAGCCGTCGCAGTCTACACATATTCTCTGTC --insert-----GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-5ʹ Protocol Step 2.5 – GEM Incubation Gel Bead Gel Bead Oligo Primer PN-2000132 P5 10x Partial Barcode Read 1N 5’-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNNNNNNNNNN-TCGTCGGCAGCGTC-3’ Linear Amplification DNA Product P5 Insert 10x Read 1N Barcode Read 2N 5ʹ-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNNNNNNNNNN-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG----insert----CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3ʹ Protocol Step 4.1 – Sample Index PCR SI-PCR Primer B PN-2000128 Forward Primer: Reverse Primer: Partial P5 P7 5’-AATGATACGGCGACCACCGAGA-3’ Sample Index N Partial Read 2N 5’- CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-GTCTCGTGGGCTCGG-3ʹ i7 Sample Index Plate N, Set A PN-3000262 Sample Index PCR Product P5 10x Read 1N Barcode Insert Read 2N Sample Index N P7 5ʹ-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNNNNNNNNNN-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG---insert---CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-NNNNNNNN-ATCTCGTATGCCGTCTTCTGCTTG-3ʹ 3ʹ-TTACTATGCCGCTGGTGGCTCTAGATGTG-NNNNNNNNNNNNNNNN-AGCAGCCGTCGCAGTCTACACATATTCTCTGTC---insert---GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-NNNNNNNN-TAGAGCATACGGCAGAAGACGAAC-5ʹ Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A 47
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