title: Illumina HiSeq 2500 paired end sequencing; ChIP-seq analysis of RNA polymerase II occupancy in wt, wee1-50 and cdc25-22 fission yeast cells
design: ChIP-seq analysis of RNA polymerase II occupancy in wt, wee1-50 and cdc25-22 fission yeast cells
library_construction_protocol: Samples were fixed in formaldehyde solution (1% final) and quenched with glycine.  After washing twice with cold PBS (phosphate-buffed saline), cells were re-suspended in lysis buffer containing proteases inhibitors and disrupted vigorously with acid-washed glass beads 8-11 times for 20 sec in a FastPrep instrument. Cells were grown in YES to an OD600 of ~ 0.8 Samples were sonicated on a Bioruptor intrument(at High setting and 6 times 5 mins with 30 secs ON/30 secs OFF). Chromatin was immunoprecipitated with antibodies against Rpb1 (ab817) or Rpb1 CTD-ser 5 (ab5408 or sigma 04-1572), which were coupled to Dynabead protein-G and protein-A and Dynabead sheep anti-mouse or rat IgG (Invitrogene). DNA was purified from immunoprecipitated samples using a MinElute Qiagen kit. Quantification of the DNA was done using QuDye dsDNA HS assay kit and quality was verified on a Bioanalyzer. Libraries were made using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (E6240S) with the indexes provided in NEBNext Multiplex Oligos for Illumina (Index Primers Set1,2 and 3).
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: 3E8(sigma 04-1572)_anti-RNA polII CTD(S5-P)