title: GSM4336652: Pol2ser5 ChIP-seq replicate 3 - KO fear; Mus musculus; ChIP-Seq
library_construction_protocol: Mouse brain tissue was homogenized in 1 ml of 1% formaldehyde for 15 min. The sample was added of 50 ml of 2.5M glycine (final concentration 125 mM), incubated at room temperature for 5min, and centrifuged at 1,000 x g for 5 min at 4°C. The pellet was collected and washed with PBS. The pellet was then resuspended in 400 ml of RIPA buffer with protease inhibitor cocktail and 0.5 mM phenylmethylsulfonyl fluoride and centrifuge at 1,000 x g for 10 min at 4°C. The lysates were sonicated to shear the DNA to lengths between 200 and 400 bp, and were centrifuged at 1,000 x g for 10 min at 4°C. In order to pre-clear the chromatin, 100 ml of Protein A beads were added to the sample and incubated for 60 min at room temperature. During preclearing step, Protein A beads were incubated with blocking solution (0.5% BSA in PBS) for 60 min at room temperature. 5 ml of antibody and 20-30 ml of Protein A beads were added into the precleared lysate and incubated for 4 hrs at 4C and mixed on a rotator during all the incubation step. Each sample was washed one-time with 0.3 M NaCl/RIPA buffer, one-time with RIPA buffer, one-time with 0.25 M LiCl and twice with TE. The beads were incubated for 8 hrs at 65C with 100 ml TE, 3 ml of 10% SDS and 3ml of Protease K in a thermocycler, and the liquid was transferred into a new tube. 100 ml of TE was added to the beads, resuspended, and the supernatant was transferred again to the tube in order to reach a final volume of 200 ml. ChIP-seq libraries were prepared for sequencing using standard Illumina protocols
attributes.tag: GEO Accession
attributes.value: GSM4336652