GSM1826467: TKS191 E14 Tip5 coverage and smoothed-peaks; Mus musculus; ChIP-Seq
Cells for ChIP were crosslinked in 1% formaldehyde for 15 min at room temperature, followed by quenching of the formaldehyde in 0.125 M glycine for 5 min. After washing the cells with PBS, they were scraped in cell lysis buffer (4% SDS, 50 mM Tris-HCl at pH 8.0, 10 mM EDTA, 100 mM NaCl, 1 mM PMSF) containing protease inhibitor (Roche). The cell lysate was incubated for 30 min at room temperature, and the crude extract was loaded on top of 8 M urea followed by ultracentrifugation overnight. The pure chromatin pellets were collected in dialysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA, 5% glycerol 0.1% Triton X-100), and dialyzed for 16 hrs at 4°C. Chromatin recovered from dialysis was fragmented to 300–800 bp by restriction enzyme digestion followed by sonication in a Bioruptor (Diagenode, high power, 30 sec on and 30 sec off for 15 cycles) in sonication buffer (0.1% SDS and 0.1% DOC in dialysis buffer). To separate insoluble components, we centrifuged the samples at 13,000 rpm for 5 min at 4°C and recovered the supernatant and pellet separately. The pellets were dissolved in sonication buffer, sonicated again for 15 cycles in a Bioruptor on high power, and centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was combined with the previous one, and a reverse-crosslinked aliquot was examined for chromatin shearing efficiency on a Bioanalyzer 2100 (Agilent Technologies Inc.). Reverse crosslinking of chromatin was done at 55°C overnight in buffer containing 1% SDS, 200 mM NaCl, 10 mM EDTA and 200 µg/ml proteinase K followed by incubation with 100 µg/ml RNase A at 37°C for 1 hr. DNA was recovered by using QIAquick PCR purification kit (Qiagen). DNA from ChIP and input chromatin fractions were sheared to an average size of approximately 300 bp using a Bioruptor UCD-200 (Diagenode) and verified on a Bioanalyzer 2100 (Agilent). For each ChIP-seq experiment, we used a custom library construction protocol. Briefly per library, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters and amplified with 15 rounds of PCR. The amplified library was selected for fragments over 200 bp in length using Agencourt AMPureXP or SPRI beads.
GEO Accession
GSM1826467