GSM7050034: h3k27ac_BALBcJ_Kupffer_Control_rep3; Mus musculus; ChIP-Seq
Mice were humanely euthanized by exposure to CO2 and liver non-parenchymal cells processed for fluorescence activated cell sorting of Kupffer cells, with modifications from published methodology (Seidman et al., 2020; Sakai et al., 2019). In brief, livers were retrograde perfused for 3 min at a rate of 5-7 ml/min through the inferior vena cava with HBSS without Ca++ or Mg++ supplemented with 0.5 mM EGTA, 0.5 mM EDTA, and 20 mM HEPES. Perfusions were then switched to 40 ml of a digestion buffer, held at 37C, comprised of HBSS with Ca++ and Mg++ supplemented with 0.033 mg/ml of Liberase TM (Roche), 20 μg/ml DNaseI (Worthington), and 20 mM HEPES. Livers were then excised, minced, and digested for an additional 20 minutes in vitro at 37C with gentle rotation in 20 ml of fresh digestion buffer. The perfusion and digestion steps were performed in the presence of 1 μM flavopiridol to offset transcriptional changes associated with digestion. After tissue digestion, cells were passed through a 70-micron cell strainer and hepatocytes removed by 2 low-speed centrifugation steps at 50 X G for 2 min. Nonparenchymal cells in the supernatant were further separated from debris by pelleting for 15 min at 600 X G in 50 ml of 20% isotonic Percoll (Sigma Aldrich) at room temperature. Cells were then washed from Percoll containing buffer and suspended in 10 ml 28% OptiPrep (Sigma Aldrich) and carefully underlaid beneath 3 ml of wash buffer. The resulting gradient was centrifuged at 1,400 X G for 25 minutes at 4C with no break and cells enriched at the interface were saved and subjected to isotonic erythrocyte lysis. Enriched non-parenchymal cells were then washed, suspended in PBS, then stained for 10 minutes with Zombie NIR (BioLegend) and purified anti-CD16/32 (93, BioLegend) to label dead cells and block Fc receptors. Cells were then immunolabeled with specific antibodies of interest, washed, and sorted using a Beckman Coulter MoFlo Astrios EQ configured with spatially separated 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers. Chromatin immunoprecipitation and sequencing libraries were generated as previously described (Texari et al., 2021) with the modifications to lysis, immunoprecipitation buffer, and washing buffer as described (Eichenfield et al., 2016; Seidman et al., 2020). In brief, FACS purified cells were fixed with 1% paraformaldehyde for 10 min at room temperature. Next, 2.625Mglycine was added to 125mM to quench fixation and cells were collected by centrifugation with the addition of 0.01% Tween-20 at 1,200 X G for 10 min at 4°C . Cells were washed once with 0.01% Tween-20 in PBS and collected by centrifugation at 1,200 XG for 10 min at 4°C . Cell pellets were then snap frozen and stored at -80C. For ChIP reactions, cell pellets were thawed on ice and lysed in 80 ml LB3 (10mMTris/HCl pH 7.5, 100mMNaCl, 1mMEDTA, 0.5mM EGTA, 0.1% deoxycholate, 0.5% sarkosyl, 1 x protease inhibitor cocktail, and 1 mM sodium butyrate). Lysate was sonicated using a Covaris for 12 cycles with the following setting: time, 60 s; duty, 5.0; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were collected and 10% Triton X-100 was added to 1% final concentration. One percent of the sonicated lysate was saved as a ChIP input. For each chromatin immunoprecipitation, aliquots of ~500,000 cells were added to 20 µl Dynabeads Protein A with 2 µg anti-H3K27ac (Active Motif) and incubated with slow rotation at 4°C overnight. The following day, beads were collected using a magnet and washed three times each with wash buffer I (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, and 1 x protease inhibitor cocktail) and wash buffer III (10 mM Tris/HCl pH 7.5, 250 mM LiCl, 1% Triton X-100, 0.7% Deoxycholate, 1 mM EDTA, and 1 x protease inhibitor cocktail). Beads were then washed twice with ice cold 10 mM Tris/HCl pH 7.5, 1 mM EDTA, 0.2% Tween-20. Sequencing libraries were prepared for ChIP products while bound to the Dynabeads Protein A as in (Texari et al., 2021). For F1 samples, dual indexed libraries were pooled for a targeted depth of 100 million reads per sample.