title: GSM6745659: Bulk ATAC-seq on FACS populations from the mouse liver (CD73 high); Mus musculus; ATAC-seq
library_construction_protocol: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, 50 mg/kg). Livers were perfused for 5 minutes with 40 mL of perfusion medium SC-1 (8 g/L Nacl, 400 mg/L KCl, 75.5 mg/L NaH2PO4, 120.5 mg/L Na2HPO4, 2,38 g/L HEPES, 350 mg/L NaHCO3, 190 mg/L EGTA, 900 mg/L D-(+)-Glucose, 1.2 mL Phenol Red solution) to remove the blood, followed by perfusion with 40 mL of SC-2 medium (8 g/L Nacl, 400 mg/L KCl, 75.5 mg/L NaH2PO4, 120.5 mg/L Na2HPO4, 2,38 g/L HEPES, 350 mg/L NaHCO3, 560 mg/L CaCl2.2H2O, 1.2 mL Phenol Red solution) containing 10 mg of collagenase P (Merck) for 5 minutes. Each lobe was dissected off and minced into small pieces in a beaker containing 39 mL SC-2 supplemented with 1 mL DNase I (Sigma-Aldrich) and 20 mg collagenase P, followed by rotating incubation for 15 min at 37°C. Hepatocytes were centrifuged for 2 min at 50 g, washed with PBS, centrifuged again for 2 min at 50 g, resuspended in 3 ml Hoechst buffer (DMEM + 10% FBS + 10 mM HEPES) and filtered through a 70 µm strainer. The protocol for hepatocyte staining was adapted from Ben-Moshe S. et al. After counting the cells on a LUNA cell counter, the concentration was adjusted to 5 million cells in 1 mL of Hoechst buffer. To determine the ploidy of hepatocytes, DNA was stained with Hoechst (Thermo Fisher Scientific) (15 µg/mL). Reserpine (5 µM) was also supplemented to prevent Hoechst expulsion from the cells. Cells were incubated for 30 min at 37°C. Hepatocytes were centrifuged for 5 min at 1,000 rpm at 4°C and the supernatant was discarded. Cells were resuspended in cold PBS in a concentration of 1 million cells in 100 µL. After spinning down (1,000 rpm for 5 min at 4°C), cells were resuspended in FACS buffer (2 mM EDTA, pH 8, and 0.5% BSA in 1x PBS) at a concentration of 1 million cells in 100 µL. Cells were stained with the following antibodies (BioLegend) at a dilution of 1:300: PE anti-mouse/human CD324 E-cadherin (catalogue no. 147304) and APC anti-mouse CD73 (catalogue no. 127210). FcX blocking solution (BioLegend catalogue no. 101319) was added at a dilution of 1:50. Cells were also stained with Alexa Fluor 488 Zombie Green (BioLegend) to enable the detection of viable cells by FACS. Zombie Green was added at a dilution of 1:500 and cells were kept in a rotator in the dark at room temperature for 15 min. Cells were sorted by FACS-Aria-Fusion (BD Biosciences) using a 100 µm nozzle. FSC-A and SSC-A were used for hepatocytes size selection. Viable cells were selected based on the Zombie Green signal. Tetraploid hepatocytes were selected based on Hoechst stain. CD73 and Ecad were used to select hepatocytes bins along the porto-central axis, obtaining 20,000-50,000 cells per bin. Omni-assay for transposase-accessible chromatin using sequencing (OmniATAC-seq) was performed as described previously. 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'. 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.