GSM3722053: SU005_Total_Post; Homo sapiens; ATAC-seq
All protocols to generate scATAC-seq data on the 10x Chromium platform, including sample prep, library prep, instrument and sequencing settings, are  available here: https://support.10xgenomics.com/single-cell-atac. The isolation, washing, and counting of nuclei suspensions were performed according to the Demonstrated Protocol: Nuclei Isolation for Single Cell ATAC Sequencing (10x Genomics). Briefly, anywhere from 100,000 to 1,000,000 cells were added to a 2 ml microcentrifuge tube and centrifuged (300 rcf for 5 min at 4°C). The supernatant was removed without disrupting the cell pallet and 100 µl chilled Lysis Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20; 0.1% Nonidet P40 Substitute; 0.01% Digitonin and 1% BSA) was added then pipette mixed 10 times. The microcentrifuge tube was then incubated on ice, with the length of time optimized for each cell type: GM12878 and A20 cell lines were 5 min; PB and BM cells were 3 min. Following lysis, 1 mL of chilled Wash Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20 and 1% BSA) was added and the resulting solution and pipette mixed 5 times. Nuclei were centrifuged (500 rcf for 5 min at 4°C) and the supernatant removed without disrupting the nuclei pellet. Based on the starting number of cells and desired final nuclei concentration, an appropriate volume of chilled Diluted Nuclei Buffer (10x Genomics; 2000153) was used to resuspend nuclei. The resulting nuclei concentration was determined using a Countess II FL Automated Cell Counter. Nuclei were then immediately used to generate single cell ATAC-seq libraries as described below. scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits User Guide (10x Genomics; CG000168 Rev A). Briefly, the desired number of nuclei were combined with ATAC Buffer (10x Genomics; 2000122) and ATAC Enzyme (10x Genomics; 2000123/2000138), to form a Transposition Mix which was then incubated for 60 min at 37°C. A Master Mix comprising of Barcoding Reagent (10x Genomics; 2000124), Reducing Agent B (10x Genomics; 2000087) and Barcoding Enzyme (10x Genomics; 2000125/2000139) was then added to the same tube as Transposed Nuclei. The resulting solution was loaded onto a Chromium Chip E (10x Genomics; 2000121) in a Chip Holder (10x Genomics; 330019). Vortexed Chromium Single Cell ATAC Gel Beads (10x Genomics; 2000132) then Partitioning Oil (10x Genomics; 220088) were also loaded onto the same Chromium Chip E before attaching a 10x Gasket (10x Genomics; 370017/3000072) and placing into on a ChromiumTM Single Cell Controller instrument (10x Genomics, Pleasanton, CA, USA). Resulting single-cell GEMs were collected at the completion of the run (~7 min) and linear amplification was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad; 1851197): 72°C for 5 min, 98°C for 30 s, cycled 12 x: 98°C for 10 s, 59°C for 30 s and 72°C for 1 min. Emulsions were coalesced using tje Recovery Agent (10x Genomics; 220016) then subjected to Dynabeads (2000048) and SPRIselect reagent (Beckman Coulter; B23318) bead clean-ups. Indexed sequencing libraries were constructed by combining the barcoded linear amplification product with a Sample Index PCR Mix comprising of SI-PCR Primer B (10x Genomics; 2000128), Amp Mix (10x Genomics; 2000047/2000103) and Chromium i7 Sample Index (10x Genomics; 3000262). Amplification was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98°C for 45 s, cycled variable amounts depending on cell load: 98°C for 20 s, 67°C for 30 s, 72°C for 20 s with a final extension of 72°C for 1 min. The barcode sequencing libraries were subjected to a final bead clean-up SPRIselect reagent and quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms; KK4824). Sequencing libraries were loaded on an Illumina sequencer with 2 × 50 paired-end kits using the following read length: 50 bp Read 1N, 8 bp i7 Index, 16 bp i5 Index and 50 bp Read 2N.
GEO Accession
GSM3722053