GSM8150685: Quiescent-Srcap_ATAC_2; Mus musculus; ATAC-seq
Cells expressing sgRNAs (BFP+) were FACS sorted in 1X PBS + 0.05% BSA, pelleted and permeabilized in 1X TD buffer containing 2 ul Tn5 enzyme (Illumina) and 0.01% Digtonin in Molecular Biology Grade water Omni-ATAC protocol: Tagmentation was performed at 37 ºC for 30 minutes with agitation at 1000 rpm, then stopped by adding EDTA 5 mM final concentration. Next, 2 uL of Proteinase K were added and the samples were incubated for 30 minutes at 40 ºC. Genomic DNA was purified with a 2X SPRI cleanup and tagmented sites were PCR-amplified with 25 uL Kappa HIFI 2X and 2.5 uL of 10 uM P5-i5-read1 and P7-i7-Read2 primers. The PCR cycling conditions are: 5 min at 72 ºC, 10 x [2 min at 98 ºC, 20 s at 98 ºC, 30s at 60 ºC, 1 minute at 72 ºC] 3 min at 72 ºC. Amplified ATAC-seq libraries were purified with a 1.6X SPRI Cleanup, quantified using the Qubit dsDNA high sensitivity assay kit, and then analysed on a Tape Station 4000. ATAC-seq libraries were sequenced at 50 million reads on a NextSeq 2000.