H3k9 14ac

ChIP and ChIP-seq were performed as previously described (30 (link),45–46 (link)). Briefly, cells were harvested and fixed with 1% formaldehyde. Chromatin was fragmented using a Bioruptor™ UCD200 sonicator (Diagenode SA, Belgium) on high power at 4°C and precipitated using antibodies against CTCF (Millipore, #07-729), H3K4me3 (Millipore, #04-745), H3K4me2 (Millipore, 07-030), H3K27Ac (Abcam, #ab4729) and H3K9/14ac (Diagenode, #C15410005). ChIP-DNA was recovered using phenol/chloroform/isoamyl alcohol and assayed by quantitative polymerase chain reaction (qPCR). ChIP primers are listed in the Supplementary Table S1. For ChIP-seq, the ChIP DNA library was prepared by ‘TruSeq ChIP Sample Preparation Kit’ (Illumina, #IP-202-1024). Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed as previously described (30 (link)). Briefly, 5 × 10⁴ cells were harvested and lysed in lysis buffer on ice. Nuclei were extracted and processed by ‘Nextera DNA Library Preparation Kit’ (Illumina). Libraries were purified with the Qiagen MinElute PCR Purification Kit. ChIP-seq and ATAC-seq libraries were sequenced using the Hiseq2500 (100PE) platform with 25M reads per sample.

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described previously [85 (link),86 (link)]. Briefly, 20 × 10⁶ cell equivalents of fragmented chromatins (containing DNA fragments < 400 bp) were immunoprecipitated with Ig isotype control or antibodies specific for histone modifications. We used the following histone antibodies in the ChIP experiments: H3K27me3 (Millipore, Billerica, MA) and H3K9/14ac (Diagenode, Denville, NJ). Immunoprecipitated DNA were purified, and then analyzed by quantitative real time PCR using primers specific for the promoter region of the mouse MCC or Diras2 gene. Primer used are: mMCC-U519F (5’- CAG GGA GGT TGG AGA GGA -3’) and mMCC-U446R (5’- AAA CAT GCC CTG CCC TTG -3’); mDiras2-U559F (5’- GCA CAT GTG ACT ACT ATT G -3’) and mDiras2-U480R (5’- AAT CTC TCC TCC CAC AAG -3’). The enrichments of each gene promoter immunoprecipitated by histone marks were quantitated relative to the input DNA [86 (link)].