GSM3929254: D7: ME22; Mus musculus; ChIP-Seq
Chromatin was isolated as described previously (Cates et al., 2018) from frozen tissue, after fixation in 1% formaldehyde and quenching in 2M glycine. Fixed tissue punches were mechanically processed by being passed through a 22-guage needle 10 times. Cells were lysed for 15 minutes (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP40, protease inhibitors), followed by a 10 minute nuclear lysis (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS, protease inhibitors).  Chromatin was sheared using a sonicator (Bioruptor, Diagenode) with 25 cyles of 30 seconds on/off. Sheared chromatin was centrigured at high speed and resuspended in RIPA buffer. Magnetic M-280 Dynabeads Sheep anti-Rabbit IgG (Thermofisher, cat #11202D) were conjugated to H3K79me2 antibody (Abcam, cat #ab3594) by rotation at 4C for 6 hours. Conjugated beads and chromatin were incubated together with rotation at 4C overnight. H3K79me2-associated DNA was washed in order with a low salt buffer (0.1% SDS, 1% Triton x100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8), a high salt buffer (0.1% SDS, 1% Triton x100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl pH 8), a LiCl buffer (150 mM LiCl, 1% NP40, 1% NaDOC, 1 mM EDTA, 10 mM Tris-HCl pH 8), and TE + 50 mM NaCl. DNA was eluted in ChIP elution buffer (1% SDS, 100 mM NaHCO3) and de-crosslinked overnight at 65 degrees C. RNA from whole tissue was prepared using the RNeasy Mini Kit (Qiagen, cat #74104) and converted to cDNA using the iScript cDNA Synthesis Kit (Bio Rad, cat #1708890) Sequencing libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB, cat #E7645) and sheared to an average size of 300-400 bp. Libraries were submitted to sequencing with Genewiz on an Illumina HISeq 4000 machine with a 2x150 bp paired-end read configuration to a minimum depth of 30M reads.
GEO
GSM3929254
GEO Accession
GSM3929254