title: GSM5963648: scATAC Alcohol hepatocytes; Mus musculus; ATAC-seq
library_construction_protocol: For snATACseq: Hepatocytes were isolated by 2-step collagenase perfusion method from pair-fed and alcohol-fed mice and counted after trypan blue staining. About 200 - 250K hepatocytes were washed with 1 ml PBS (0.04% BSA) and were lysed for 15 min on ice using 1X lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween 20, 0.1% NP40, 0.01% Digitonin). Nuclei were washed with 1 ml of wash buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween 20) and resuspended in 10 - 30 ml of wash buffer. A fraction of nuclei was stained with DAPI and the number of nuclei was counted from the merged images of bright field and DAPI-positive. . Transposition reaction targeting approximately 3,000 - 8,000 nuclei per sample was performed by mixing 10 μL of 1X Tn5 Transposition enzyme (10X Genomics) with 5uL of the diluted nuclei suspension and incubating the reaction at 50°C for 1h. The tagmented nuclei were partitioned into Gel Bead-In Emulsions (GEMs) and barcoded by unique molecular identifiers (UMIs) using the Chromium Next GEM Single Cell ATAC library & Gel Bead kit and the C1 Chromium Instrument following the manufacturer's instructions (10X Genomics).  scATAC-seq libraries were amplified by PCR and indexed using the Chromium i7 Sample Index kit (10X Genomics). The size of purified library DNA was determined by Fragment Analyzer (Advanced Analytical Technologies) and the enrichment of accessible region was determined by the fold difference between positive and negative genomic loci using real-time PCR. The following primers were used: AT-MDusp6, forward: GGCTTATCCGGAGCGGAAAT; AT-MDusp6, reverse: GGCTGGAACAGGTTGTGTTG; AT-MVmn2r17, forward: TCCCCTTTACTGTTTTCCTCTAC; AT-MVmn2r17, reverse: GGATTGATGAGGAAACAGCCTC. Two libraries per lane on an Illumina Hiseq4000 instrument were sequenced to 51 base pairs from both ends in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility.