GSM2178376: P28_input; Mus musculus; ChIP-Seq
Flow-sorted cells were lysed using lysis buffer and sonicated using Covaris. Cellular debris was removed by centrigugation, and clear chromatin solution was reconstituted to RIPA buffer. Pre-cleared chromatin solution was then incubated with anti-H3K4me3 (ab8580, Abcam, MA, USA), anti-H3K27me3 (07-449, Millipore, MA, USA) or normal IgG (12-370, Millipore) with constant rotation at 4°C overnight. Thoroughly washed immunoprecipitation complexes were then digested, and the cross-link was reversed by incubation in 50 l of TE, pH 8 supplemented with 0.3% SDS and 1 mg/ml proteinase K at 65 °C for 6 hrs. The carried-over RNA was digested by 1 hr incubation at 37°C with RNase cocktail (Ambion, Life Technologies), and the immunoprecipitated DNA was then purified using Agencourt AMPure XP beads (Beckman Coulter, CA, USA). Libraries were prepared using TruSeq DNA Sample Preparation kit v2 (Illumina, FC-121-2001; FC-121-2002) according to Illumina's instructions with the following modifications. 1.  As amount of total ChIP DNA obtained from each ChIP experiment is estimated to be only 400 pg to 1 ng, entire ChIP DNA was used for ChIP-seq library construction Instead of 1 ug DNA as recommended by Illumina. 2. For adapter ligation, 1/100 dilution of adapter was used to avoid having access adapter-adapter dimer. 3. After adapter ligation, double-sided SPRI technique (200-500 nt fragments were selected with 0.55x left /0.75x right) was used for size-selection in order to minimize a sample loss. 4. 18 cycles of PCR amplification were performed on all ChIP-seq libraries.
GEO Accession
GSM2178376