GSM1841045: H3K27me3_J1_2iVitC_D15_rep2; Mus musculus; ChIP-Seq
Cells were cross-linked directly in the culture plate with 1% formaldehyde (culture medium supplemented with 1% formaldehyde, 0.015M NaCl, 0.15mM EDTA, 0.075mM EGTA, 15mM Hepes pH 8). Formaldehyde was quenched with 0.125M glycine and cells were washed in PBS and pelleted. Cells were then rotated at 4°C for 10 minutes in buffer 1 (Hepes-KOH pH 7.5 50mM, NaCl 140mM, EDTA pH 8.0 1mM, glycerol 10% NP-40 0.5%, Triton X-100 0.25% and the protease inhibitors: PMSF 1mM, Aprotinin 10μg/ml, leupeptin 1μg/ml and pepstatin1μg/ml), resuspended and rotated at room temperature for 10 minutes in buffer 2 (NaCl 200mM, EDTA pH 8.0 1mM, EGTA pH 8.0 0.5mM and 10mM Tris pH 8 and the same protease inhibitors as buffer 1) and then resuspended in buffer 3 (EDTA pH 8.0 1mM, EGTA pH 8.0 0.5mM, Tris pH8 10mM, N-lauroyl-sarcosine 0.5% and the protease inhibitors as buffer 1). Chromatin was sonicated with a Bioruptor (Diagenode) to reach a fragment size around 200bp. Chromatin corresponding to 10μg of DNA was incubated overnight at 4°C with 3-5μg of antibody in incubation buffer (buffer 3 supplemented with 0.5 volume of 3% Triton, 0.3% sodium deoxycholate, 15mM EDTA and protease inhibitors as in buffer 1). 5% of chromatin extracts were taken aside for inputs. Chromatin bound to the antibody was recovered using Active Motif Protein G Agarose Columns. Briefly, the antibody-chromatin mix was incubated in the column for 4 hours, washed eight times with modified RIPA buffer (Hepes pH7.6 50mM, EDTA pH 8.0 10mM, sodium deoxycholate 0.7%, NP-40 1%, LiCl 500mM, PMSF 1 mM, 1μg/ml leupeptin and 1μg/ml pepstatin), and washed one last time with TE-NaCl (50mM Tris pH 8.0, 10mM EDTA, 50mM NaCl). Chromatin was eluted with pre-warmed TE-SDS (50mM Tris pH 8.0, 10mM EDTA, 1% SDS). ChIP-enriched sample and inputs were then reverse cross-linked at 65°C overnight and treated with RNase A and proteinase K. DNA was extracted with phenol/chloroform/isoamyl alcohol, precipitated with glycogen in sodium acetate and ethanol and finally resuspended in TE. 20ng of ChIP- or input-DNA were used for ChIP-seq. Remaining large DNA fragment were first eliminated using SPRIselect beads (Beckman Coulter) and libraries were prepared using the TruSeq ChIP Sample Prep kit from Illumina.
GEO Accession
GSM1841045