title: GSM8667809: Vibhakar_1520_two_4; Homo sapiens; ATAC-seq
library_construction_protocol: Human MB samples were collected and viably banked at the time of surgery at Children's Colorado with consent (COM-IRB 95-500) over a 10-year period. Samples were processed to obtain single nuclei for single-cell Multiome sequencing according to the 10X Genomics (Pleasanton, CA) protocol. Briefly, a rice grain size of cyropreserved tumor tissue was homogenized in chilled 0.1X Lysis Buffer with a pellet pestle.  Lysis buffer(10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2, 0.1%Tween-20, 0.1% NP-40, 0.01% Digitonin, 1% BSA, 1mM DTT, 1U/mL RNase Inhibitor) and Lysis Dilution buffer(10mM Tris-HCl(pH7.4), 10mM NaCl, 3mM MgCl2, 1%BSA, 1mM DTT, 1U/mL RNAse inhibitor).  Samples were incubated on ice for 5 min, mixed, then iced again 10 min. Samples are washed with 500mL of chilled wash buffer (10mM Tris-HCl(pH7.4), 10mM NaCl, 3mM MgCl2, 1%BSA, 0.1% Tween-20, 1mM DTT, 1U/mL RNase inhibitor) and passed through a 40mM strainer twice. The samples were centrifuged at 500 rcf for 5 min at 4°C. The nuclei pellet was then washed twice with 1 mL of chilled wash buffer. Nuclei were counted on the Countess II FL automated cell counter, and nuclei were diluted in nuclei buffer and submitted for sequencing. The Chromium Next GEM Chip J Single Cell Kit (10xGenomics), Dual Index Kit TT set A, and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle were used for sample preparation and library sequencing on the Chromium Library construction followed the 10x genomics Chromium Next GEM Single Cell Multiome. ATAC + Gene Expression user guide. ATAC LibraryConstruction Action Item 10x PN Preparation & Handling Storage Equilibrate to Room Temperature Sample Index 3000427- −20°C Plate N, Set A Place on Ice 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® Prepare 80% Ethanol Prepare 10 ml for 8 reactions Step 5 ATAC Library Construction 5.1 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 Total - 57.5 253 506 b. Transfer 40 µl pre-amplified sample from step 4.3p to a new tube strip (35 µl of the remaining sample volume will be used for cDNA Amplification and the rest can be stored at −20°C long term for generating additional libraries). Add 57.5 µl Sample Index PCR Mix to the sample. Pipette mix and centrifuge briefly. c. Add 2.5 µl of an individual 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 hh:mm:ss 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 Targeted Nuclei Recovery Total Cycles ≤2,000 9 2,001-6,000 8 STOP e. Store at 4°C for up to 72 h or proceed to the next step.  A Single Cell Gene Expression library is generated using a fixed proportion (10 µl, 25%) of the total cDNA (40 µl) obtained at step 6.2n. The complexity of this library will be comparable to one generated using a higher proportion (>25%) of the cDNA. The remaining proportion (30 µl, 75%) of the cDNA may be stored at 4°C for up to 72 h or at −20°C for longer-term storage (up to 4 weeks). Correlation: cDNA input & Library Complexity Library Complexity (Median genes/UMI's) cDNA Volume (µl) (% total cDNA) Recommended 10 µl (25%) 20 µl (50%) 30 µl (75%) 40 µl (100%) Note that irrespective of the total cDNA yield (ng), which may vary based on cell type, targeted nuclei recovery etc., this protocol has been optimized for a broad range of input mass (ng), as shown in the example below. The total number of SI PCR cycles (step 7.5d) should be optimized based on carrying forward a fixed proportion (10 µl, 25%) of the total cDNA yield calculated during Post cDNA Amplification QC & Quantification (step 6.3). Example: Library Construction Input Mass & SI PCR Cycles Cell Type Targeted Nuclei Recovery Total cDNA Yield (ng) cDNA Input into Fragmentation SI PCR Cycle Number Volume (µl) Mass (ng) Low High RNA Content 150 ng 10 µl 37.5 ng 14 High 400 ng 10 µl 100 ng 13 Low RNA Content Low High 1 ng 10 µl 0.25 ng 16 100 ng 10 µl 25 ng 14 Click to TOC Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F 60Step 7 7.1 Fragmentation, End Repair & A-tailing Click to TOC ! Gene Expression Library Construction a. Prepare a thermal cycler with the following incubation protocol. Lid Temperature Reaction Volume Run Time hh:mm:ss 65°C 50 µl ~35 min Step Temperature Time Pre-cool block Pre-cool block prior to preparing the Fragmentation Mix 4°C Hold Fragmentation 32°C 00:05:00 End Repair & A-tailing 65°C 00:30:00 Hold 4°C Hold b. Vortex Fragmentation Buffer. Verify there is no precipitate. c. Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly. Fragmentation Mix Add reagents in the order listed PN 1X (μl) 4X + 10% (μl) 8X + 10% (μl) Fragmentation Buffer 2000091 5 22 44 Fragmentation Enzyme 2000090/ 2000104 10 44 88 Total - 15 66 132 d. Transfer ONLY 10 µl purified cDNA sample from cDNA Cleanup (step 6.2n) to a tube strip. Note that only 10 µl (25%) cDNA sample is sufficient for generating 3ʹ Gene Expression library. The remaining 30 µl (75%) cDNA sample can be stored at 4°C for up to 72 h or at −20°C for up to 4 weeks for generating additional Gene Expression libraries. e. Add 25 µl Buffer EB to each sample. f. Add 15 µl Fragmentation Mix to each sample. g. Pipette mix 15x (pipette set to 35 µl) on ice. Centrifuge briefly. h. Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the