GSM1934417: ChIP-seq NPC C-20; Homo sapiens; ChIP-Seq
Cell pellets were suspended in 1 ml of Cyto lysis buffer, mixed briefly, and incubated on ice for 10-15min with occasional inversion every 2min.  Cells were pelletted for 5min at 3500rpm, 40C, Supernatant discarded, and the nuclear pellets were resuspended in 500ul of nuclear lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-Cl, pH8.1, 1Xprotease inhibitor cocktail) and incubated on ice for 10min.  The pellets were sonicated seven times for 10 sec each at the maximum setting (Branson, Sonifer cell disruptor 185). The lystate was cleared by centrifugation at 14000 rpm for 10 min in 4C and the supernatant was collected in 15ml tube. The supernatant was diluted 1:10 with the dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-Cl, pH8.1, 1Xprotease inhibitor cocktail) to yield the soluble chromatin fraction, 100ug-200ug of which was used for each ChIP reaction. Lystate was pre-cleared by adding 20ul of Protein A/G beads followed by rotation for 1 hour at 4C. 5ug of specific antibodies were applied to the supernatant and incubated overnight at 4C. 50ul of Protein A&G magnetic beads were blocked with (1xPBS+5mg/ml BSA-fraction V) and incubated at 4C for another 4 hours. Magnetic beads were harvested on a magnetic stand and beads wished sequentially at 4C, twice in 0.7 ml of TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.1, 150 mM NaCl), twice in TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.1, 500 mM NaCl), twice in Buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, pH8.0, 10 mM Tris-HCl, pH8.1), and finally, twice with 1ml of TE buffer at 4C.  Eluted beads were twice washed with 150ul of elution buffer (TE buffer, 1%SDS) by votexing at 70C, 1000rpm for 10 min. The eluents were heated at 65C overnight to reverse formaldehyde-induced crosslinks. For input sample, 150-300ul of soluble chromatin was also heated at 65C overnight. DNA fragments were purified with a QIAquick Spin PCR purification Kit (Qiagen, CA). ChIP-seq libraries were constructed by using the TruSeq ChIP Sample Prep Kit (Illumina).
GEO Accession
GSM1934417