GSM8041681: RXRα_Chipmentation_rep2; Mus musculus; OTHER
Peritoneal exudate were isolated by peritoneal lavage, fluorescent antibodies staining, and flow sorting by CD11b hi F4/80 hi cells. Cells were fixed with 1% fresh formaldehyde (Thermo Fisher Scientific) for 10 minutes at room temperature with rotation, followed by quenching using 0.125 M glycine for 5 minutes. After washing with ice-cold PBS supplemented with cOmplete protease inhibitor (Roche), cells were resuspended in lysis buffer (10 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.25% SDS, 1x protease inhibitor), and sonicated on the Covaris M220 ultra-sonicator to obtain 200-300 bp DNA fragments using the following settings: peak incident power 50, duty factor 10%, cycles per burst 200, time 8 minutes. Keep 2 µL of supernatant from two replicates as the input DNA at -20 °C. The remaining supernatant was incubated overnight at 4°C on a rotator with 3 µg of anti-RXRα (D6H10) Rabbit mAb (#3085, Cell Signaling), or Rabbit IgG (#2729, Cell Signaling) antibodies pre-coupled to Protein A Dynabeads (Invitrogen). The beads were washed twice sequentially on a pre-cold magnet (Invitrogen) using RIPA-LS wash buffer (10mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), RIPA-HS wash buffer (10mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), and RIPA-LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) and followed by a wash with 10 mM Tris-HCl pH 8.0. After washing, the beads were then resuspended in 25 µL tagmentation reaction mix containing 1 µL Tn5 transposase (Illumina), and incubated at 37°C for 5 minutes. After removing the tagmentation reaction mix, beads were washed twice with RIPA-LS wash buffer and once with TE buffer (10 mM Tris-HCl pH 8.0, 500 mM EDTA), followed by elution with 48 µL elution buffer (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.4% SDS) containing 2 µL Proteinase K (NEB) at 55°C for 1 hour and 65°C for 8 hours for de-crosslinking. The input DNA was also incubated with tagmentation reaction mix, and de-crosslinked with Proteinase K. 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.