GSM5493536: High concentration rep 4; Homo sapiens; ATAC-seq
Single cell-containing collagen was carefully collected by pipette tip into microcentrifuge tubes, and an equal volume of 5,000 U (μmol/min) collagenase type 7 (Worthington) dissolved in DPBS (Gibco) was added into each tube for incubation for 15 minutes at 37°C. After digestion, cells were spun down at 4°C, and then resuspended into freezing media (10% DMSO, 20% FBS, and 70% culture media) for freeze-down. Viable frozen cells were thawed rapidly at 37°C, and then slowly diluted using DMEM supplemented with 10% FBS until the diluted volume reached at least 10-fold of the initial volume. If needed, samples were concentrated by centrifuging at 500 RCF for 5 minutes, followed by removal of supernatant. Omni-ATAC protocol (Corces, M. R. et al., Nature Methods, 2017) was followed, with the following modifications: a cell count of up to 25,000 per sample was used, and cells were washed twice in ATAC-RSB (resuspension buffer) containing 0.1% Tween-20 prior to lysis and transposition. A reaction volume of 25 μL was used for transposition. After the transposition, half of the reaction product was amplified by PCR using a total of 11 cycles without qPCR testing, followed by SPRIselect bead (Beckman Coulter) clean-up (2:1 bead ratio). All the buffers (RSB-Tween, lysis buffer, and TD buffer) were made fresh before use. After the library preparation, samples were quantified by a Qubit (Thermo Fisher) and the size distribution was determined by a Fragment Analyzer (Agilent). If necessary, a digital PCR assay (Bio-Rad QX200) was used to accurately quantify the sequenceable molarity. The libraries were then sequenced on an Illumina HiSeqX at Novogene with 2x150 paired-end reads. ATAC-seq. Omni-ATAC (Corces, M. R. et al., Nature Methods, 2017)