GSM5267241: ATAC_WT_P7_rep2; Homo sapiens; OTHER
ATAC-seq was performed as published (Buenrostro et al., 2013). 10,000-50,000 cells were used per biological replicate where possible; if cells numbered less than 10,000 an optimized low cell number protocol was used. Cells were spun at 500g for 10 min, then lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl 3 mM MgCl2, 0.1% IGEPAL CA-630). Transposition was then performed with Tn5 transposase (Nextera, Illumina) for 30 min at 37ÅãC following the reaction volumes as published. DNA was purified using the Qiagen MinElute kit following the manufacturer's instructions, then eluted in 20μl of elution buffer; samples could be stored at this point at -20ÅãC. Libraries were amplified and barcoded using the NEBNext 2xMastermix kit with custom primers as published (Buenrostro et al., 2013). ATAC-seq library profiles were visualized using the D1000 tape on the Agilent Tapestation to ensure correct tagmentation. Libraries were quantified using the universal library quantification kit (KAPA Biosystems) prior to massively parallel sequencing using a standard Illumina protocol (NextSeq High Output v2 75 cycle sequencing kit). The low cell number protocol uses proportionally less lysis buffer and Tn5 transposase (Corces et al., 2017). After spinning at 500g for 10 min and removing the supernatant, cells were permeabilised and tagmented with Dig-transposition buffer (25 μL 2x TD buffer [Illumina], 2.5 μL Tn5 Transposase, 0.5 μL 1% digitonin and 22 μL H2O) for 30 min. After the transposition step, samples were processed as per larger cell numbers. Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013. ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). The libraries were quantified using the universal library quantification kit (KAPA Biosystems).