GSM1820991
GSM1820991: V5 ChIP-seq in NS5 Neural Stem Cells; Mus musculus; ChIP-Seq
Extraction protocol For each ChIP 1.5*10^8 NSCs were used. NS-5 NSCs expressing no V5-tag were used for the control experiment. NSCs were crosslinked with 2 mM disuccinimidyl glutarate (DSG, Thermo Fisher Scientific) for 45 min at RT, washed three times (PBS + 1mM PMSF) and then crosslinked for 10min with 1% formaldehyde. This reaction was quenched by addition of 1/20 volume of 2.5M glycine for 10min at RT. Cells were washed twice with cold 1x PBS+1mM PMSF, collected by centrifugation, and cell pellets frozen with N2(l). Cells were resuspended in LB1 (50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton-X-100 . After 10 minutes incubation, cells were pelleted by centrifugation and resuspended in LB2 (10 mM Tris-HCl, pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA. After 10 minutes incubation, cells were pelleted and resuspended in freshly prepared LB3 (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine and sonicated on a Soniprep 150(MSE). For quality control of the sonication, it was verified that the DNA fragment size was between 100 and 1000bp. V5-Agarose beads were blocked 1 hr with 0.5 mg/ml BSA. The chromatin was incubated overnight with the blocked V5-Agarose beads. The following day the beads were washed 5 times with RIPA buffer and eluted with elution buffer (50mM Tris-HCl pH8, 10mM EDTA and 1% SDS) for 30 minutes. Samples were de-cross-linked overnight at 65C and treated the following day with proteinase K for one hour at 45C. DNA was extracted by phenol/chloroform/isoamyl extraction and precipitated with ethanol and resuspended in water. The library was prepared according to the illumina protocol. Briefly 10 ng of DNA ChIP material was end-repaired, ligated to adapters, size selected on gel (200 bp +-25 bp range) and PCR amplified using Phusion polymerase with 30 seconds incubation at 98 ᴼC, 18 cycles of 10 second 98 ᴼC , 30 seconds 65 ᴼC, 30 seconds 72ᴼC. Followed with 5 minutes of final extension at 72 ᴼC. Cluster generation was performed using the Illumina HiSeq 2000 platform.
GEO
GSM1820991
GEO Accession
GSM1820991