title: GSM7025900: Input_Chipmentation_for_Aire_rep1; Mus musculus; OTHER
library_construction_protocol: Cells were fixed with 1% formaldehyde (28906, Thermo Fisher Scientific) for 10 min at room temperature with rotation, followed by quenching using 0.125M glycine for 5 min at room temperature. After washing using ice-cold PBS supplemented with protease inhibitor (Complete EDTA-free protease inhibitor cocktail, Roche), cells were resuspended in lysis buffer (10mM Tris-HCl pH 8.0, 140mM NaCl, 1mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100) supplemented with protease inhibitor on ice for 10 min. Chromatin was then sonicated on the Covaris M220 ultrasonicator with the following settings: peak incident power 50, duty factor 10%, cycles per burst 200, time 5 min. Lysate was centrifuged for 10 min at 14,000g at 4°C. An “input” sample was preserved from the supernatant. The rest of the supernatant was incubated with 2 ug of anti-Aire (14-5934-82, eBioscience) or anti-NELF (ab170104, abcam) antibody precoupled to Protein A Dynabeads (Invitrogen) on a rotator overnight at 4°C. On the following day, beads were washed twice sequentially on a pre-cold magnet (Invitrogen) using wash buffer 1 (10mM Tris-HCl pH 8.0, 140mM NaCl, 1mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), wash buffer 2 (10mM Tris-HCl pH 8.0, 500mM NaCl, 1mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), wash buffer 3 (10mM Tris-HCl pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) and 10mM Tris-HCl pH 8.0. Beads were then resuspended in 25uL tagmentation reaction mix with 1uL Tn5 transposase (Illumina) added, and were incubated at 37°C for 5 min. After removing the tagmentation reaction mix, beads were washed twice with wash buffer 1 and once with 1 x TE. They were then incubated with 50uL elution buffer (10mM Tris-HCl pH 8.0, 300mM NaCl, 5mM EDTA, 0.4% SDS) containing 2uL Proteinase K (NEB) at 55°C for 1h and 65°C for 8h to de-crosslink. DNA was purified using MinElute Reaction Cleanup Kit (QIAGEN). Libraries were indexed and amplified using the NEBNext High-Fidelity 2x PCR Master Mix (M0541S, NEB) and ATAC-seq primers, followed by purification using AMPure XP beads.