GSM9540769: H3k4me3 control R2 mESC; Mus musculus; ChIP-Seq
Following centrifugation the cell pellet was lysed in 5 ml of lysis buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% Triton-X-100, 1x protease inhibitor cocktail, 1 mM PMSF), then washed in 5 ml of buffer (5 mM HEPES pH 8.0, 85 mM KCl, 1x protease inhibitor cocktail, 1 mM PMSF), before being resuspended in buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA, 1x Protease inhibitor cocktail, 1 mM PMSF) and snap-frozen on dry ice. Once the samples were thawed, sonication was carried out with 5 cycles of 30 seconds on/off (Bioruptor Plus (Diagenode), low intensity setting). Following centrifugation, 10% of the sample was removed for input. The input sample was treated with proteinase K (NEB) overnight at 65°C and purified using Zymo ChIP and concentrate, and used to check for sonication efficiency. The remaining sample was precleared with a 1:1 mix of protein-A and protein-G Dynabeads (pre-equilibrated in PBS 0.1% BSA) for 1 hour at 4°C. The pre-cleared sample was then mixed with the antibody overnight at 4°C, before 1:1 mix of protein-A and protein-G Dynabeads (pre-equilibrated in PBS 0.1% BSA) were added for 2 hours. The beads were then washed 3 times in FA/SDS solution (50 mM HEPES KOH pH 7.5, 150 mM NaCl, 1% Triton-X-100, 0.1% Na deoxycholate, 1x Protease inhibitor cocktail, 1 mM PMSF) with the last wash being performed for 10 minutes at 4°C. The beads were then washed once in WB buffer (10 mM Tris-HCl pH 8, 0.25 M LiCl, 1 mM EDTA, 0.5% NP40, 0.5% Na-deoxycholate), once in TE (Tris/EDTA) buffer pH 8.0, before being eluted in elution buffer (25 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% SDS) at 65°C for 25 minutes. The samples were treated with RNase (NEB) for 30 minutes at 37°C before being reverse crosslinked overnight with Proteinase K at 65°C, and finally purified with Zymo ChIP clean and concentrate. For ChIP-seq, libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina accordingly.