GSM2572839: rep2_AI_K27_input; Homo sapiens; ChIP-Seq
Androgen-dependent prostate cancer and its androgen-independent derivative cell lines was used to do the ChIP-Seq library construction. Approximately 1 × 107 cells were fixed with 1% formaldehyd, and were resuspended in 1 mL SDS Lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1). DNA was sonicated to 200 – 500 b. After centrifugation, we took 60ul supernatant and diluted tenfold with ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). The lysate was then incubated with anti-EZH2 antibody(source: Millipore, cat# 17-662, lot# 2366583) or anti-H3K27me3 antibody(source: Millipore,cat#07-449,lot# 2382150) at 4 ◦ C overnight. The immunuprecipitates were further incubated with Pierce™ Protein A/G Magnetic Beads (Thermo, 88803) for 2 hours at 4 oC. After applying to magnet and removing the supernatants, we washed beads once with Low Salt Immune Complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl) and added 1 ml of buffer to each tube, rotated at 4 ℃ for 5 min; we then placed the tube on the DynaMag-2 magnet for 30 s, then discarded supernatant. We repeated washing first with High Salt Immune Complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl) and LiCl Immune Complex wash buffer(0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1) and second with TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for a total of 5 washes. The immunoprecipitates were eluted from the beads with elution buffer (50 nM Tris 8.0, 10 mM EDTA and 1% SDS) and incubated at 65 oC overnight. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. ChIP-Seq was performed by using ThruPLEX® DNA-seq kit (R400427; Rubicon Genomics) according to manufacturer’s instructions. The PCR products corresponding to 300-500 bps were gel purified, quantified and stored at -80 oC until used for sequencing. ChIP-Seq sequencing was performed on NextSeq500 platform with 151 bp paired-end sequencing.