title: GSM6659531: atac_C57_Kupffer_Control_rep1; Mus musculus; ATAC-seq
library_construction_protocol: 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. Transposase reactions and sequencing libraries were generated as described previously (Buenrostro et al., 2013; Sakai et al., 2019; Seidman et al., 2020) using 25,000 to 50,000 FACS purified Kupffer cells. Tagmented DNA was cleaned up using Zymo ChIP Clean & Concentrate columns and PCR amplified for 14 cycles using barcoding primers. Libraries were size selected to 175-225 bp using gel excision and purified as described (Texari et al., 2021). For F1 samples, dual indexed libraries were pooled for a targeted depth of 100 million reads per sample.