GSM1820996
GSM1820996: Smad4 ChIP-seq in NS5 Neural Stem Cells; Mus musculus; ChIP-Seq
For each ChIP 1.5*10^8 NSCs were used. As a control, the same experiment was performed in parallel using goat IgG. 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 whether the DNA fragment size was between 100 and 1000bp. The DNA concentration was measured in order to take a volume of chromatin solution containing 300µg DNA per ChIP. Per sample, 150µl prot-G Dynabead solution (Life Technologies) was used. Beads were blocked for 1hr at RT with 0.5mg/ml BSA and 0.2mg/ml sonicated salmon sperm DNA in IP buffer (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 1% Triton X-100) After blocking, the beads were washed 3 times in IP buffer. To lower unspecific binding, the chromatin was incubated with beads without the presence of antibody, rotating for 30 min at 4 C. The beads were discarded and the 'precleared' chromatin was incubated overnight at 4 C with 15µg of antibody. The next day, 150µl of blocked beads was added to this mixture and rotated for 3hrs at 4 C. The supernatant was discarded and the beads were washed subsequently by rotation for 10min for each wash: 2x low salt buffer(20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 1% Triton X-100), 2x high salt buffer (20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 1%Triton X-100, 0.1% SDS), 2x LiCl buffer (10mM Tris-HCl pH8, 250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% deoxycholate) and finally twice in TE (10 mM Tris-HCl pH8, 1mM EDTA). Immune complexes were eluted in 210ul elution buffer (1% SDS, 0.1 M NaHCO3)  for 1 h at 65 °C while softly shaking. The crosslinks were reversed by incubation overnight at 65C. The following day, the samples were treated with proteinase K for one hour at 45C. DNA was extracted by phenol/chloroform/isoamyl extraction and precipitated with ethanol. 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
GSM1820996
GEO Accession
GSM1820996