title: GSM2719694: H3_D2_rep3_ChIPSeq; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: For ChIP-seq,chromatin immunoprecipitation was performed as described (Ercan et al. 2007; Landt et al. 2012).  Chromatin extract was incubated with H3 antibody, H3K4me3 antibodies overnight at 4°C.  Antibodies used were prescreened for specificity using dot blots.  The optimal amounts of antibodies used were determined by titration in preliminary experiment with ChIP-qPCR.  5-15 ng precipitated DNA from each sample was used for illumina sequencing library preparation.  DNA from ChIP was first end-repaired to generate blunt end, followed by adding single adenine base for adaptor ligation. Ligation product with adaptor was size selected and amplified by PCR with primers targeting adaptor. Worms at L3,adult day 2, and adult day 12 stages were washed with ice cold M9 three times.  Worm pellets were stored at -80°C before chromatin immunoprecipitation and RNA extraction. For ChIP-seq, chromatin immunoprecipitation was performed as described (Ercan et al. 2007; Landt et al. 2012).  Worm pellet was grinded with mortar/pestle and cross-linked with 1% formaldehyde in PBS at room temperature for 10 min.  Worm fragments were collected by spinning at 3,000 g for 5 min and resuspended in FA buffer followed by sonication with Bioruptor. For ribo-RNAseq, total RNA was extracted from worms harvested at the same stages at adult day 2 and day 12 as ChIP-seq sample preparation.