title: GSM2825910: H3K36me3_M2; Homo sapiens; ChIP-Seq
library_construction_protocol: First, cells were fixed for 5 min at 37°C by adding room temperature Crosslinking Buffer to culture medium (to final concentrations of 50 mM TrisHCl pH 8, 100 mM NaCl, 1 mM EDTA, 1% formaldehyde). Fixation was topped by adding Glycine to a final concentration of 250 mM and incubates 5 min at room temperature. After two washes of ice-cold PBS, cells were scraped twice in 30 ml and 15 ml PBS. Cells were pelleted for 10 min at 4000 rpm 4°C, washed in ice-cold PBS and resuspended in 10 ml Cell Lysis Buffer (10 mM TrisHCl pH 8, 25 mM KCl, 0.5% NP-40) supplemented with AEBSF. After incubating 10 min on ice, lysate was passed 10 times through a 20G needle and incubated again for 10 min on ice. Nuclei were pelleted for 15 min at 3500 rpm 4°C and resuspended in 3.4 ml Sonication Buffer (50 mM TrisHCl pH 8, 10 mM EDTA, 0.1% SDS) supplemented with AEBSF. Nuclei were then divided in 2 tubes and sonicated for 20 min at 4°C on the Diagenode Bioruptor Pico according to manufacturer's instructions. Finally, poorly sonicated chromatin was pelleted for 10 min at 16 000g and supernatant was recovered and added to blocked Protein A Dynabeads IP beads for pre-clearing of chromatin. After incubating for 3 hours at 4°C on a rotating wheel, 200 µl were taken as Input. The rest of the chromatin was incubated overnight at 4°C on a rotating wheel with IP antibodies: H3(pan) (Abcam ab1791), H3K36me3 (Abcam ab9050-100), H3K4me3 (Diagenode pAb-003-050), H3K27me3 (Active Motif 39155) or H3K27ac (Abcam ab4729). The next day, 50µl blocked IP beads were added to the chromatin and incubated 4 hours at 4°C on a rotating wheel. They were then washed twice in FA150 buffer (50 mM HEPES pH 7.4, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.1% NA-Deoxycholate, 0.1% SDS), twice in FA500 (50 mM HEPES pH 7.4, 1 mM EDTA, 1% Triton X-100, 500 mM NaCl, 0.1% Na-Deoxycholate, 0.1% SDS) and once in TE (10 mM TrisHCl pH 8, 1 mM EDTA). To elute chromatin, beads were resuspended in 100 µl ChIP Elution Buffer (50 mM TrisHCl pH 7.4, 10 mM EDTAs 1% SDS) and incubated for 1 hour at 65°C with 650 rpm shaking. Supernatant was then recovered, NaCl was added to a final concentration of 200 mM and incubated overnight in a 65°C waterbath to remove crosslinks. The next day, samples were treated with RNase for one hour at 37°C and with proteinase K for one hour at 45°C. DNA was then purified using Qiagen PCR Purification columns and quantified using Qubit dsDNA HS kit. ChIP-seq libraries were prepared with TruSeq ChIP Sample Prep Kit (IP-202-1012, Illumina)
attributes.tag: GEO Accession
attributes.value: GSM2825910