SMCHD1 ChIP-seq in hTERT-RPE1. The human SMCHD1-specific mouse monoclonal antibody was raised against an SMCHD1 fragment (amino acids 1645–2006), expressed in and purified from E. coli as a His6-fusion protein.
Sequenced DNA Library
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).