GSM8281059: WT_Treg (Foxp3 ChIP-seq input); Mus musculus; ChIP-Seq
Cells were fixed with 1 % formaldehyde for 30 minutes at room temperature. Cells were Lysed with lysis buffer 1 (50 mM HEPES, 140 mM sodium chroride, 1 mM EDTA, 10 % glycerol, 0.5% NP-40, 0.25% Triton X-100, 5mM DTT with protease inhibitor) for 10 minutes on ice, lysis buffer 2 (10 mM Tris-HCl, 200 mM sodium chloride, 1 mM EDTA, 0.5 mM EGTA with protease inhibitor) for 10 minutes on ice, and suspended in lysis buffer 3 (10 mM Tris-HCl, 300 mM sodium chloride, 1mM EDTA, 0.5 mM EGTA, 0.1 % sodium deoxycholate, 0.5 % N-lauroylsacrosine with protease inhibitor). Lysed cells were sonicated in lysis buffer 3. The cell extract was incubated overnight at 4 ℃ with 2-4 ug of anti-BATF (clone: D7C5, CST) or anti-Foxp3 antibodies (in-house) and then with Dynabeads protein G (Dynal) for 4 hours at 4 ℃. DNA-protein complexes bound to Dynabeads were washed with low salt buffer (0.1 % SDS, 1 % Triton-X, 2 mM EDTA, 20mM Tris-HCl, 150mM sodium chloride) once, high salt buffer (0.1 % SDS, 1 % Triton-X, 2 mM EDTA, 20 mM Tris-HCl, 500mM sodium chloride) twice, RIPA buffer (50 mM HEPES, 500 mM LiCl, 1 mM EDTA, 1 % NP-40, 0.7 % sodium deoxycholate) fourth, and TE buffer containing 50 mM sodium chloride once. The DNA-protein complexes were eluted from the Dynabeads by heating for 10 minutes at 65 ℃ with vortexing in elution buffer (50 mM Tris-HCl, 10 mM EDTA, 1 % SDS) and crosslinking was reversed by overnight incubation at 65℃. The immunoprecipitated DNA and whole-cell extract input DNA were treated with RNase A and proteinase K, purified using MinElute PCR Purification Kit (QIAGEN). NEBNext ChIP-Seq Library Prep Reagent Set for Illumina and NEBNext Ultra II DNA Library Prep Kit for Illumina