GSM4064083: Vok.NLE.H3K9me3; Nomascus leucogenys; ChIP-Seq
We fixed 5x106 cells per ChIP assay, with 1% formaldehyde for 5min on ice and then quenched fixation by adding glycine to a final concentration of 0.1M. After washing the cells twice with cold 1X PBS, we lysed the fixed cells at a concentration of 3x106cells/100ul in Lysis buffer (0.1% SDS, 0.5% Triton X-100, 20mM Tris-HCl pH=8.0, 150mM NaCl, 1x Proteinase inhibitor (Roche)) for 5min on ice. Using the Bioruptor Pico sonicator (Diagenode), we sheared lysates in 1.5mL tubes with 7 cycles (30sec on/off). We spun the sonicated lysates at max speed for 10min at 4°C to remove cell debris and then rotated them for 2hr at 4°C with 20ul ChIP-grade Protein A/G Magnetic Beads (Pierce) to preclear the lysate. From each chromatin preparation, we set aside a 1% volume aliquot as chromatin input, and the rest we divided equally into each histone mark ChIP reactions. We diluted the reactions to 1.6x106cells/100ul with Lysis buffer and then added the corresponding antibodies in the following amounts: 2ug H3K4me1 (ab8895, Abcam), 1ug H3K4me3 (ab8580, Abcam), 1ug H3K27ac (ab4729, Abcam), 2ug H3K27me3 (39155, Active Motif) and 2ug H3K9me3 (ab9263, Abcam), and let reactions rotate overnight at 4°C. Next, we added 20ul Pierce ChIP-grade Protein A/G Magnetic Beads (Pierce) and let samples rotate for 2hrs at 4°C. We then washed the beads consecutively (rotating at 4°C) with TBST buffer (3 times for 15min), Lysis buffer (once for 1hr), 1xTE pH=8 (once for 1hr, then once for 10min). Following washes, we eluted chromatin in 250ul fresh Elution buffer (1% SDS, 0.1M NaHCO3). We incubated the ChIP and input samples overnight at 65°C in presence of NaCl to reverse crosslinks and then digested them consecutively with 8ug RNAse A (30 minutes at 37°C) and 80ug Proteinase K (2 hour at 55°C). Lastly, we purified the samples using traditional phenol:chloroform extraction and ethanol precipitation. We quantified all samples using the Qubit dsDNA High Sensitivity kit (Thermo Fisher Scientific). All sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) for 1-5ng of starting material and without size selection. The Qubit dsDNA High Sensitivity kit (Thermo Fisher Scientific) and Agilent Bioanalyzer 2100 were used to QC the libraries.
GEO
GSM4064083
GEO Accession
GSM4064083