title: GSM1604438: Rph1- Young (S2Y, Exp 1) H3; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation was performed as described previously from 4 X 10^8 cells (W. Dang et al., Nature 459, 802 (Jun 11, 2009)). Briefly, cross-linked cells were lyzed by bead beating in FA lysis buffer (50mM HEPES-KOH, pH 7.5, 500mM NaCl, 1mM EDTA, 0.1% Triton X-100) and the chromatin sheared using a Diagenode Bioruptor or Covaris Ultrasonicator to achieve an average size of <500bp. Equal aliquots of sonicated chromatin were used per immunoprecipitation (IP) reaction (H3K36me3 (Active Motif 61101) and H3 (Abcam ab1791)) and 10% of the amount was saved as input. IP was performed using protein A Dynabeads (Life Technologies). ChIP-qPCR was performed against standards made from a dilution series of an input DNA mix. Enrichment was ascertained by calculating signal over IgG. To ensure that we were getting H3K4me3 and H3K36me3 enrichment at 5’ ends and 3’ ends respectively, ChIP-qPCR validation was performed with 5’ and 3’ end primer sets over long genes before library preparation for ChIP-seq. Primer sequences are available upon request. For ChIP-seq, 1ng of DNA from IP and input was used to prepare libraries using the Microplex library preparation kit (Diagenode). Twelve multiplexed libraries were sequenced in a single lane on the Illumina HiSeq or NextSeq 500 platform.
attributes.tag: GEO Accession
attributes.value: GSM1604438