GSM2197651: Pdd1p ChIP from new MACs of PDD1 KO rescued with wild-type PDD1; Tetrahymena thermophila; ChIP-Seq
Chromatin immunoprecipitation was performed with the new MACs only when their purity was above 80%. Chromatin from the new MACs was sheared into 150-500 bp fragments (peak ~300 bp) in Sonication buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1% SDS, 1x complete protease inhibitor cocktail, 1 mM PMSF) using a probe sonicator. For ChIP, 20 mg of Protein A Sepharose CL-4B beads (GE Healthcare) was washed with TE twice and with ChIP buffer (25 mM Tris-HCl pH 8.0, 167 mM NaCl, 5 mM EDTA, 0.05% SDS, 1% Triton X-100, 1x complete protease inhibitor cocktail, 1 mM PMSF) 3 times and was then pre-incubated with 90 µl of ChIP buffer containing 10 µl of 10 mg/ml BSA (NEB) (total ~200 µl suspension) at 4°C for 1 h. The fragmented chromatin from 2 million new MACs in Sonication buffer was diluted with an equal volume of 2x ChIP dilution buffer (334 mM NaCl, 2% Triton X-100, 1x complete protease inhibitor cocktail), adjusted to 1 ml with ChIP buffer and incubated with a 25 µl suspension of the beads at 4°C for 2 h for the pre-absorption. To couple an antibody to the beads, the 10 µl suspension of the beads pre-incubated with BSA was incubated with an antibody (20 µg anti-H3K9me3, 30 µg anti-H3K27me3 or 5 µg anti-Pdd1p) in 0.5 ml of ChIP buffer at 4°C for 2 h and was then washed with ChIP buffer 3 times. The pre-absorbed chromatin was incubated with the antibody-coupled beads at 4°C overnight. The beads were washed once with Low-salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) and twice each with High-salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), LiCl wash buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40) and TE. The immunoprecipitated chromatin was eluted with Elution buffer (0.1 M NaHCO3, 1% SDS). For cross-linking reversal, the NaCl concentration of the sample was adjusted to 200 mM, and the sample was incubated at 65°C for ~12 h. The DNA was extracted, and DNA libraries were generated from the immunoprecipitated DNA and 20 ng of the fragmented chromatin DNA without immunoprecipitation (Input) using the NEBNext DNA Library Prep Reagent Set for Illumina (NEB) and the KAPA Real-Time Library Amplification Kit (KAPA Biosystems). The libraries were sequenced using the HiSeq2000 platform (Illumina) with 50-nt single-end reads.