GSM1463440: Mat_Bcell_H3K4me3; Mus musculus; ChIP-Seq
Cells were fixed with 1% formaldehyde in PBS (20 ml vol./ ~1 × 108 cells) for 10 min, followed by quenching with 2.5 M glycine for 5 min. Cells were lysed for 10 min in 1 ml lysis buffer A (10 mM HEPES, pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25 % Triton X-100 + PI). The supernatant was discarded and the nuclei were incubated for 10 min in buffer B (10 mM HEPES, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01 % Triton X-100 + PI). Next, the nuclei were suspended in chromatin lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5 % SDS) for 30 min. Sonication was performed using Bioruptor Next Gen (Diagenode) at high output intensity for 25 cycles (30on/30off). Finally the collected supernatant was diluted 5x with chromatin dilution buffer (250 mM NaCl, 1.67 % Triton X-100). The chromatin (25-50 µg) was subjected to the IP with the above mentioned antibodies and incubated overnight. The IPs were incubated with either protein A or G coupled to magnetic beads for 1 h. The beads were washed with the following buffers, twice with low salt (0.10% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 15 mM NaCl), high salt (0.10% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 50 mM NaCl), LiCl (250 mM LiCl, 1 M EDTA, 10 mM Tris pH8.0, 1% NP-40, 1% Na Deoxychholate) and once with TE (20 mM Tris-HCl, pH8.9, 2 mM EDTA). The protein-DNA complexes were eluted from the beads with 500 µl elution buffer (10mM Tris-HCl, pH7.5, 1mM EDTA, 1% SDS, 100 mM NaHCO3) and reverse cross-linked overnight at 65°C with Proteinase K. ChIPed DNA was isolated by phenol/ chloroform extraction and ethanol precipitated. 10ng of ChIPed DNA were processed for Illumina HiSeq analyzer according to the manufacturer's protocol. Total RNA was extracted from 3 different cultures of HSCs, Pro B cells and LPS activated mature B cells using RNeasy Mini Kit #Qiagen 74104. Every cell culture is a pool of cells extracted from 3 mice. 10 ng of RNA were used for library preparation (ScriptSeq v2 RNA-Seq Library Preparation Kit) and sequenced according to the Illumina protocol. Sequencing libraries were prepared using bar-coded adapters following standard Illumina library preparation protocol. Four samples carrying different barcodes were pooled at equal molar ratios and subjected for sequencing on Illumina HiSeq 2000 sequencer according to Illumina standards.