GSM1940100: OSKM Day 10 Jmjd3+/+ FLAG overexpression anti-H3K27me3; Mus musculus; ChIP-Seq
Cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and then add glycine at 0.125M to quench formaldehyde. Cells were rinsed twice with cold PBS, then lysed in ChIP Lysis Buffer A (50mM HEPES-KOH pH7.3; 140mM NaCl; 1mM EDTA pH8.0; 10% glycerol; 0.5% NP-40; 0.25% Triton-100; protease inhibitor cocktail) for 10 min at 4 ℃. Samples were centrifuged at 1,400g for 5min at 4 ℃, discard the supernatant and re-suspend the samples with ChIP Lysis Buffer B (1% SDS; 50mM Tris-HCl pH8.0; 10mM EDTA; protease inhibitor cocktail) for 10min at 4 ℃. Lysates were sonicated into about 150-300 bp fragments using Bioruptor (Diagenode), and centrifuged at 14,500 g at 4 ℃ for 10 minutes to discard insoluble pellets, and the supernatant was diluted ten times with ChIP IP Buffer (0.01% SDS, 1% Triton-100, 2mM EDTA, 50mM Tris-HCl pH8.0, 150mM NaCl, protease inhibitor cocktail), then incubated with specific antibody overnight at 4 ℃, and add protein A/G Dynabeads (Invitrogen) to capture specific immunoprecipitates. Wash the beads 3 times with low salt buffer (0.1% SDS, 1% Triton-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 150mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 500mM NaCl), once with LiCl buffer 0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH8.1), and twice with TE buffer (10mM Tris-HCl, 1mM EDTA pH8.0). After washing, add 200ul 10% Chelex-100 and 2ul of Proteinase K (PK,20mg/ml) to incubate in a Mixing Block (BIOER) at 56 ℃ for 30 min, followed by boiling in water for 10 min, then centrifuged at 5,000 rpm for 5 min, and transfer the supernatant to a new tube. Antibodies used were: anti-H3K27me3 (Millipore, 07-449). Drosophila S2 cells were cross-linked in the same way mentioned above, and lysed in ChIP Lysis Buffer A for 10 min, centrifuged, and re-suspended with ChIP Lysis Buffer B, then mixed together with reprogramming mouse cells with the ratio of 1:2. Once Drosophila S2 cells and reprogramming cells were combined, the sample was treated as a single sample throughout the ChIP-seq experiment. After sonication, samples were centrifuged at 13.200 rpm at 4 ℃ for 10 min, the supernatant was diluted twice with ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl, protease inhibitor cocktail) and dialyzed with Slide-A-Lyzer Dialysis cassette (Thermo Scientific, 66380) to reduce the high level of SDS followed with incubation with antibody and Dynabeads. Washed beads were eluted with fresh Elution Buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA, 1.0% SDS) at 56 ℃ for 30 minutes. Centrifuge and incubate the supernatant at 65 4 ℃ for 8-16 h to reverse crosslink the immunoprecipitation DNA. After incubated with RNase A and PK, DNA was purified with phenol:chloroform:isoamyl alcohol (P:C:IA). Standard Illumina protocols
GEO
GSM1940100
GEO Accession
GSM1940100