GSM1820995
GSM1820995: IgG control ChIP-seq in NS5 Neural Stem Cells for Olig2; 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 rabbit IgG. NSCs were washed 3 times with PBS, crosslinked with 1/10 volume of fresh 11% buffered formaldehyde solution for 12 min., quenched with 1/20 volume of 2.5M Glycine for 5 min, washed with ice-cold PBS and cell pellets frozen with N2(l) and resuspended and washed 2 times in ice-cold cell lysis buffer (10mM Tris-Cl pH 7.5, 10mM NaCl, 3 mM MgCl2, 0.5% NP40) and cell pellet resuspended in lysis buffer with 1mM CaCl2 and 4% NP40 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, that was next diluted 1 to 5 with ChIP dilution buffer (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 1% Triton X-100 and 1x CEF protease inhibitor). Equilibration of preblocked protein G beads (Millipore) was performed by washing them 3x with 1 ml of ChIP dilution buffer. To lower unspecific binding, the diluted 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, 60µl of 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, 5min for each wash: 1x ChIP dilution buffer, 1x high salt buffer (20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 1%Triton X-100, 0.1% SDS),1x LiCl buffer (10mM Tris-HCl pH8, 250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% deoxycholate) and twice in TE (10 mM Tris-HCl pH8, 1mM EDTA). Elution was performed two times by incubation in 250 l of pre-warmed (37C) freshly prepared elution buffer (0.1M NaHCO3, 1% SDS) for 15 minutes at RT. Supernatants were combined and centrifuged to get rid of beads. 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
GSM1820995
GEO Accession
GSM1820995