Ion Torrent Proton sequencing; H3K4me1 and H3K27ac ChIP-seq in Ring1a KO and Ring1a/b dKO mouse ES cells
H3K4me1 and H3K27ac ChIP-seq in Ring1a KO and Ring1a/b dKO mouse ES cells
Ring1A−/−;Ring1Bfl/fl;Rosa26::CreERT2 mESCs (Ring1A-KO) were cultured as described previously in Endoh et al. Dishes were coated with 0.1 % gelatine and irradiated mouse embryonic fibroblasts (MEFs). For harvesting, ESCs were trypsinized and pre-plated twice for 30 min to remove contaminating feeder cells. Conditional deletion of Ring1B in Ring1A-KO cells was carried out by addition of 800 nM tamoxifen for 48 h as previously described in Endoh et al. Ring1A-KO and Ring1A/B-dKO mESCs (2 x 108) were fixed in ChIP fix buffer (1% formaldehyde, 5 μM EGTA, 10 μM EDTA, 1 mM NaCl, and 0.5 mM HEPES in PBS) for 10 min at RT. Fixation was stopped by glycine (final concentration 125 mM). Cells were washed with PBS, buffer A (10 mM Hepes pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100) and buffer B (10 mM Hepes pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). Cell extracts were lysed in lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 1% SDS, 0.5% deoxycholate complete protease inhibitor (Roche)) for 30 min on ice. Sonication was performed using a BioRupter sonicator (Diagenode) to obtain an average DNA fragment size of 300 bp. Chromatin was diluted with ChIP dilution buffer (25 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.5% deoxycolate complete protease inhibitor (Roche)). Dynabeads® Protein G beads were blocked for 1 hr at 4°C with 1 mg/ml BSA and 1 mg/ml yeast tRNA (AM7119 Life Technologies,). For each IP 150 µg of chromatin and 5 µg of antibodies H3K4me1 (ab8895 Abcam) and H3K27ac (ab4729 Abcam) were used, for input samples the chromatin was directly taken to the DNA purification step. Chromatin was pre-cleared with blocked beads for 1 hr at 4°C. Chromatin was incubated with antibodies overnight at 4°C with rotation. Protein-antibody complexes were immuno-precipitated by addition of beads for 2 hr. Complexes were washed twice with wash buffer A (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP40, 1 mM EDTA), once with wash buffer B (50 mM Tris pH8.0, 500 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP40, 1 mM EDTA), once with wash buffer C (50 mM Tris pH8.0, 250 mM LiCl, 0.5% deoxycholate, 1% NP40, 1 mM EDTA) and once with TE. Samples were treated with RNase A and Proteinase K and cross-links were reversed overnight. DNA was purified using a ChIP DNA clean and concentrator column (Zymo Research). Libraries were prepared using NEB Next Fast DNA Fragmentation and library preperation set for Ion Torrent kit (E6285S) following manufacturer’s instructions. Briefly, 40 ng of ChIP or input DNA was used for library generation. Libraries were size-selected for 250 bp fragments using 2% E-gel Size Select agarose gel (Life Technologies) and were amplified with 5 PCR cycles.
Experimental Factor: phenotype
tamoxifen-mediated knockout of Ring1b on Ring1a-/- background
Experimental Factor: immunoprecipitate
H3K27ac