title: GSM6094615: ABC_TLR9WT.ATAC.rep3; Mus musculus; ATAC-seq
library_construction_protocol: The protocol was adapted from Corces, et al. Resuspension buffer (RSB) was made at a final concentration of 10 mM Tris-HCl pH 7.5 (Thermo Fisher #15567027), 3 mM MgCl2 (Thermo Fisher #AM9530G), and 2 mM NaCl (Thermo Fisher #AM9760G) in molecular grade H20. 20,000-50,000 viable cells were pelleted in low-bind 1.5 ml microcentrifuge tubes (Eppendorf # 022431021), and washed once with PBS supplemented with 1% fetalplex and protease inhibitor (Sigma #P8340, stock at 100x). Supernatant was vacuum aspirated, and 50 µl of cold lysis buffer containing 0.1% IGEPAL CA-360 (Sigma #I8896, 0.01% Digitonin (Promega #G9441) , and 0.1% Tween-20 (Sigma #11332465001) in RSB was added to each sample by gently pipetting. Samples were incubated on ice for 3 minutes to isolate nuclei, then washed with 950 ul of 0.1% Tween-20 in RSB and pelleted at 500 x g for 10 minutes at 4⁰ C. Supernatant was aspirated using p1000 and p200 pipets, followed by addition of 50 ul transposition mix containing 25 ul TD buffer, 2.5 ul TDE1 Tagment DNA Eznyme (Illumina #20034197), 16.5 µl PBS, 0.01% Digitonin, 1% Tween-20, brought to 50 µl with molecular grade H20. Samples were incubated at 37⁰ C for 30 minutes, with mixing every 10 minutes by flicking the tube. Transposed DNA was then purified using the Qiagen MinElute PCR purification kit (Qiagen #28004). To generate libraries, adapters (IDT for Illumina Nextera DNA Unique Dual Indexes, Illumina, #20027215) were added during amplification using 2X Phusion High Fidelity Mastermix with HF Buffer (ThermoFisher #F531L).  Libraries were cleaned up with Zymo DNA Clean & Concentrator-5 columns (Zymo Research #D4004) and assessed with a Qubit Flex Fluorometer (ThermoFisher $Q33327) and a TapeStation 4200 (Agilent #G2991BA).