H3K9me3 ChIP-seq in hTERT-RPE1 treated with HBiX1 siRNA
Chromatin immunoprecipitation (ChIP) was performed as described previously (Kimura et al., 2008), with minor modifications. hTERT-RPE1 cells (2.5x10^6 cells in 90-mm dishes or six-well plates for combination with siRNA transfection) were fixed in medium containing 5% formaldehyde at room temperature for 5 min. Chromatin was sheared to an average size of ~200 bp using a Bioruptor USD-250 bath sonicator (Cosmo-Bio; 24 sonications of 15 s with 30-s intervals at 250 W) and cleared by centrifugation (16,100xg for 10 min at 4 degree_C). The chromatin was then incubated with antimicromouse IgG magnetic beads (Invitrogen; Dynabeads M-280; 50 microl) preincubated with 1 microg of purified monoclonal mouse IgG or 10 microl of hybridoma culture supernatant for 2 h, followed by washing and de-cross-linking in 200 microl ChIP direct elution buffer. DNA was recovered from the remaining immunoprecipitated samples by proteinase K and RNase A treatments, followed by phenol-chloroform extraction and ethanol precipitation. BioProject of this experiment was changed from PRJDB2066 to PRJDB2065.
ChIP-seq libraries were prepared according to the manufacturer's instructions (Illumina). Briefly, immunoprecipitated DNA was end-repaired using a mix of Klenow DNA polymerase, T4 DNA polymerase and T4 polynucleotide kinase (NEB), tailed with an 'A' base using Klenow Fragment 3'-5' exo minus (NEB), and ligated with the Illumina single-end adaptor, using a DNA ligation Kit (TaKaRa). Adaptor-ligated fragments of approximately 300 bp were purified using the E-gel SizeSelect system (Life Technologies), and were subjected to 18 cycles of PCR amplification using KOD FX polymerase (TOYOBO). To remove the remaining PCR primers, the amplified products were further purified using AMPure XP Kits (Beckman Coulter). Libraries were quantified using a BioAnalyzer 2100 with a High Sensitivity DNA Kit (Agilent).