GSM7518534: Bulk ATAC-seq on Hepa16; Mus musculus; OTHER
The HepG2, Hepa1-6 and AML12 cell lines were purchased from ATCC (HB-8065, CRK-1830 and CRL-2254, respectively). HepG2 cells were cultured in Eagle's Minimum Essential Medium (EMEM, Thermo Fisher Scientific), Hepa1-6 cells were clustered in Dulbecco's Modified Eagle's Medium (DMEM), and AML12 in DMEM-F12 medium. All media were supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 50 µg/mL penicillin/streptomycin (Thermo Fisher Scientific).  Cell cultures were kept at 37°C, with 5% CO2. Omni-assay for transposase-accessible chromatin using sequencing (OmniATAC-seq) was performed as described previously. Briefly, 50,000 cells obtained after FACS were resuspended in 50 µL of cold ATAC-seq resuspension buffer (RSB; 10 mM TrisHCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water) containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 min. After lysis, 1 mL of ATAC-seq RSB containing 0.1% Tween-20 was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 500 g in a pre-chilled (4°C) fixed-angle centrifuge. Supernatant was removed and nuclei were resuspended in 50 µL of transposition mix (25 µL 2x TD buffer, 2.5 µL transposase (Nextera Tn5 transposase, Illumina), 16.5 µL PBS, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, and 5 µL water) by pipetting up and down six times. Transposition reactions were incubated at 37°C for 30 min in a thermoblock. Reactions were cleaned-up by MinElute (Qiagen). Transposed DNA was amplified with primers 'i5_Indexing_For' and 'i7_Indexing_Rev' (Supplementary Table 4). Number of PCR cycles was based on quantitative real-time PCR as described previously. All libraries were sequenced on a NextSeq2000 instrument (Illumina) with the following sequencing parameters: 51 bp read 1 – 8 bp index 1 – 8 bp index 2 – 51 bp read 2.