Anti ehmt1

Normal and EHMT1^+/‐ mutant human dermal fibroblasts were lysed in 4× Laemmli buffer (250 mm Tris (pH 6.8), 20% glycerol, 8% SDS, 0.0025% bromophenol blue, 1 mm β‐mercaptoethanol), collected by scraping with a rubber policeman and then sonicated for 10s. Samples were boiled at 95 ^⁰C for 5 min then subjected to 14% SDS polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes and probed with the following primary antibodies: anti‐H3K9me2 (Cell Signaling Technology, Danvers, MA, USA), anti‐total Histone H3 (Abcam, Cambridge, MA, USA) in 3% bovine serum albumin in TBST, or anti‐EHMT1 (Abcam, Cambridge, MA, USA) in 5% milk. Anti‐mouse or anti‐rabbit secondary antibodies (Millipore, Danvers, MA, USA) were incubated on the membranes, after three successive washes with TBST, for 1 h at room temperature, followed by detection of bands with ECL (Pierce, Rockford, IL, USA). Quantification of bands was done using ImageJ (Rasband).

For the preparation of chromatin immunoprecipitation sequencing (ChIP-seq), cells were fixed with 1% formaldehyde and then lysed by the ChIP lysis buffer. Chromatin was then sheared into approximately 200–300 bp fragments (∼500–800 bp for ChIP-qPCR) using Bioruptor Sonicator (Diagenode). IP was carried out using antibodies including anti-V5 (Invitrogen, 46-1157), anti-H3K9me2 (Abcam, ab1220), and anti-EHMT1 (Abcam, ab63161). DNA libraries were constructed using the SMARTer ThruPLEX DNA-Seq Prep Kit (Takara Bio). Next-generation sequencing (51 nt, single-end) was performed using the Illumina NextSeq 2000. ChIP-seq reads were mapped to the hg19 human reference genome and peak calling was performed using MACS2.