title: GSM5100089: Atac-seq - IM uninfected 1; Mus musculus; ATAC-seq
library_construction_protocol: AM and IM were sorted from lung single cell suspensions prepared as described previously (Pisu et al, 2020) using a BD FACSAria sorter. Cells were sorted in PBS containing 0.04% BSA and processed for ATAC-seq library preparation immediately. FACS-sorted cells were spun down at 500g for 5 min, followed by a wash using 50 mL of cold 1× PBS and centrifugation at 500g for 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) and immediately after lysis, nuclei were spun at 500g for 10 min. Following the nuclei prep, the pellet was resuspended in the transposase reaction mix (25 μl 2× TD buffer, 2.5 μl transposase (Illumina) and 22.5 μl nuclease-free water). The transposition reaction was carried out for 30 min at 37 °C. Directly following transposition, the sample was purified using a Qiagen MinElute kit. Then, we amplified library fragments using 1× NEB next PCR master mix and 1.25 μM of custom Nextera PCR primers using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. The libraries were purified using a Qiagen PCR cleanup kit in 20 μl. Libraries were amplified for a total of 10–13 cycles and were subjected to high-throughput sequencing using the Illumina HiSeq 2500 Sequencer (pair-end).