title: GSM8790317: ATAC-seq KO_2; Mus musculus; ATAC-seq
library_construction_protocol: Cells were kept on ice, washed twice in ice-cold PBS without Ca2+/Mg2+ and centrifuged for 5 minutes at 1300 rpm. Cells were resuspended in one volume of ice-cold PBS before adding four volumes of lysis buffer (12.5 mM Tris, pH 7.4; 45 mM KCl; 6.25 mM MgCl2; 375 mM sucrose; 0.125% Nonidet P-40; 1 tablet of complete [EDTA free] protease inhibitor mixture [Roche]/50 mL). After 10 minutes of incubation on ice, lysed cells were centrifuged at 4°C for 7 minutes at 500g. The nuclei pellet was resuspended in a tenth of the original lysis volume in wash buffer (10 mM Tris, pH 7.4; 60 mM KCl; 15 mM NaCl; 5 mM MgCl2; 300 mM sucrose). Then, wash buffer was added to half of the lysis volume and nuclei were centrifuged at 4°C for 7 minutes at 500g. Washed nuclei were counted, and 50,000 nuclei were used for the transposase reaction. 5 × 10^4 nuclei were directly treated with Tn5 tranposase at 37°C for 30 min. After the enzymatic reaction, the DNA was purified by Mini-Elute PCR Purification columns (Qiagen) and enriched by 12 PCR cycles. The library was recovered from PCR by GeneRead Purification columns (Qiagen) and double-strand sequenced on Illumina NovaSeq 6000 S4 PE100. A total of 112 to 132 × 10^6 reads/sample were obtained. For footprinting analysis, libraries were re-sequenced to allow increased sequencing depth, and an additional 103 to 149 × 10^6 reads/sample were obtained.