BioProject

SAFB has roles in gene repression, and we asked whether PRC2 was involved in this process Overall design: For H3K27me3 and total H3 ChIPs, The day before sonication, 25μL of protein A/G agarose beads (Santa Cruz sc-2003) were washed 3 times in block solution (0. More...

SAFB has roles in gene repression, and we asked whether PRC2 was involved in this process Overall design: For H3K27me3 and total H3 ChIPs, The day before sonication, 25μL of protein A/G agarose beads (Santa Cruz sc-2003) were washed 3 times in block solution (0.5% BSA in 1xPBS) before being resuspended in 300μL blocking solution. 10μL per 10 million cells of antibody (Abcam mouse monoclonal ab6002) was added, then beads and antibody conjugated via rotation overnight at 4°C. On the day of sonication, 10 million ESCs crosslinked with 0.6% formaldehyde were re-suspended in lysis buffer 1 (50mM HEPES pH 7.3, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and 1x PIC (PIC; Sigma Product #P8340) incubated for 10 minutes at 4C, and then incubated with lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, and 1x PIC) for 10 minutes at room temperature. For H3K27me3 ChIPs, cells were re-suspended in lysis buffer 3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA pH8.0, 0.5 mM EGTA pH 8.0, 0.1% Na-deoxycholate, 0.5% N-lauroyl sarcosine, and 1x PIC) and then sonicated. ChIPs were then performed by incubating sonicated cell lysates at a concentration of 20 million cells/1ml of lysis buffer 3 containing 1% Triton X-100 with pre-conjugated with H3K27me3 antibody/agarose beads overnight at 4˙C. After overnight H3K27me3 ChIP, beads were washed 5x in RIPA buffer (50 mM HEPES pH 7.3, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Na-Deoxycholate) for 5 minutes each and then once in TE. To elute the DNA, beads were re-suspended in Elution buffer (50mM Tris pH 8.0, 10mM EDTA, and 1% SDS) and placed on a 65°C heat block for 17 minutes with frequent vortexing. Crosslinks were reversed overnight at 65°C, eluates were incubated with Proteinase K and RNase A, and DNA was extracted with phenol/chloroform and precipitated with ethanol. DNA was prepared for sequencing on the Illumina platform using Next Reagents (NEB) and Agencourt AMPure XP beads (Beckman Coulter). Less...