title: GSM1874092: Forebrain_Input_control_2; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq: Cells and tissues were cross-linked with 1% formaldehyde for 15 min. Glycine (0.625 M) was added to quench excess formaldehyde. Cells were washed in cold PBS and spun down (3,000 r.p.m. (800g) for 10 min at 4 °C; Kubota 3500). The pellet was  resuspended  in FA lysis buffer and incubated for 15minutes (0.25% Triton X-100, 10 mM EDTA, 10 mM Tris HCl [pH 8.0], 100 mM NaCl, Roche 1X cOmplete protease inhibitor). Nuclei were extracted (3,000 r.p.m. for 10 min at 4 °C; Kubota 3500) and resuspended in 300 μl SDS lysis buffer (1% SDS, 1% Triton X-100, 2 mM EDTA, 50 mM HEPES-KOH [pH 7.5], 0.1% Na dodecyl sulfate, Roche 1X cOmplete protease inhibitor). After 15 min incubation, chromatin was fragmented to an average size of 200–300 bp (Bioruptor Next gen, Diagenode). Cellular debris was removed by centrifuging at 15,000 r.p.m. at 4 °C (Kubota 3500). Nuclear lysates were diluted (1:10) with a dilution buffer (1% Triton X-100, 2 mM EDTA, 50 mM HEPES-KOH [pH 7.5], 0.1% Na dodecyl sulfate, Roche 1X cOmplete protease inhibitor).   Chromatin immunoprecipitation (ChIP)  was done coupled to 50 μl protein G Dynabeads (Invitrogen) overnight. The antibodies used for ChIP were : H3K4me2 (Abcam ab32356, lot 780430)), H3K4me1(Abcam:ab8895, lot 718019), H3K27ac( Abcam ab4729, lot 509313), H3K27me3 ( Millipore 07-449, lot DAM1588246 ), PolII ( Abcam ab5131, lot 536530) , H2BK20ac ( Abcam ab52988, lot 527003). After ChIP overnight, beads were washed, and protein-DNA complexes were eluted with  elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8]).  Protease treatment and de-cross-linking was done at 65 °C overnight., DNA was extracted using phenol/chloroform and purified by ethanol precipitation. ChIP material was used for library preparation using the Biooscientific library preparation kit with the following modification. The provided adapters were diluted down 40-fold in order to prevent adapter dimer formation that would affect enrichment. Libraries were then enriched for 15 cycles with PFX polymerase (Invitrogen) followed by gel size selection (300-500bp).
attributes.tag: GEO Accession
attributes.value: GSM1874092