title: GSM4512324: ATAC_KN-RM_rep1; Mus musculus; ATAC-seq
library_construction_protocol: Hepatic macrophages: 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 (Mederacke et al., 2015; Muse et al., 2018; Seki et al., 2007). 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. Peripheral monocytes: Mice were humanely euthanized by exposure to CO2. Blood was collected from mice via cardiac puncture into K3EDTA tubes and subjected to RBC lysis (eBioscience). Cells were pelleted by centrifugation at 350 X G for 10 minutes at 4°C and 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. Next, cells were stained in wash buffer for an additional 20 minutes with the antibodies listed in the Key Resource Table. Stained cells were washed twice and strained through a 30 μm strainer, then subjected to cell sorting using a Beckman Coulter MoFlo Astrios EQ configured with 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers. Each cell population was hierarchically gated using Beckman Coulter Summit software. Ly6CHi monocytes were defined as CD45PosCD11bHiCD115PosCD19NegCD90.2NegLy6GNegLy6CHi. Ly6CHi monocytes were further restricted to single particles by comparing height and area side scatter pulses, and dead cells were excluded by detecting the integration of the live/dead dye (Zombie NIR). Approximately 50,000 sorted cells were washed once with PBS and once with 10mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% Igepal by centrifugation at 500 X G at 4C. Cells were then suspended in 50 μl reaction buffer comprised of 25 μl Tagment DNA buffer, 2.5 μl Tagment DNA enzyme, and 22.5 μl nuclease free water, using reagents sourced from Illumina Nextera DNA Library Prep Kit (Buenrostro et al., 2013). Transposase reactions were carried out at 37C for 30 minutes and immediately DNA was purified using Zymo ChIP Clean & Concentrate columns. Resulting DNA was PCR amplified for 14 cycles using barcoding primers and resulting libraries were size selected by gel excision to 175-225 bp as described in (Link et al., 2018). Library DNA was purified, and single end sequenced using a HiSeq 4000 or a NextSeq 500.
attributes.tag: GEO Accession
attributes.value: GSM4512324