GSM1202003: ChIP-seq_WT_Rpb1_I; Schizosaccharomyces pombe; ChIP-Seq
100 ml cultures grown to 1-2 x 107 cells/ml were crosslinked (1% formaldehyde, 30 min) and lysed by bead beating.  The chromatin fraction was isolated and sheared to 200-500 bp fragments using a Bioruptor sonicator (Diagenode) or Sonicator 3000 (Misonix).  Immunoprecipitations (IPs) were performed overnight at 4°C with 1 μg of anti-H3 (ab1791; Abcam), 2 μg of anti-H2B (ab1970; Abcam), 3 μg of anti-H3K36me3 (ab9050; Abcam), 5 μg of anti-H3K4me3 (ab8580; Abcam), 5 μL of anti-Rpb1 (8WG16; Covance), 5 μg of anti-HA (ab9110; Abcam), or 5 μL of anti-Myc (9E10; Santa Cruz Biotechnology).  IPs were coupled to 50 μL of protein-G-sepharose beads (GE Healthcare Life Sciences) at 4°C for 4 hours.  Beads were washed and eluted; eluate was reverse-crosslinked overnight at 65°C and incubated with proteinase K and glycogen for 2 hours at 37°C.  DNA was purified by phenol chloroform extraction and precipitated in ethanol overnight at -20°C. Between 1-10 ng of ChIP DNA were processed for each ChIP-seq library.  DNA was end repaired with T4 DNA Polymerase (Invitrogen), T4 PNK (NEB) and DNA Pol I, Large Klenow fragment (Invitrogen), and an 'A' base was added using Klenow 3' to 5' exo minus (NEB).  1 pmol of barcoded adaptors were ligated on with T4 DNA ligase (Roche) and the products were PCR amplified with Phusion DNA Polymerase (NEB) and size selected by purification on 2% agarose-EX E-gels (Invitrogen) for fragments between 200-600 bp.  Library size and concentration was determined by Bioanalyzer or Tapestation analysis (Agilent) and libraries were pooled equimolar amounts with up to 26 in each sequencing lane.  Pooled libraries were gel purified twice, followed by column purification on MinElute columns (Qiagen).  At least 10 nM were submitted for analysis on the Illumina Hi-Seq at Tufts University Core Facility.