title: GSM1291079: small RNA seq of SNPC-4(RNAi) of staged young adults; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: For all ChIP-seq samples, worms  were crosslinked in 2% formaldehyde in M9 buffer for 30 minutes rotating at room temperature.  Crosslinking was quenched with 1M Tris pH 7.5, followed by two washes with M9, and one wash with FA buffer containing protease inhibitors.  Final Triton X-100 concentration in all FA buffers used was either 1% (ChIP-seq data used in the tissue-specific analysis) or 5% (ChIP-seq data sets showing the developmental time course).  Worm pellets were flash-frozen in liquid nitrogen and stored at -80˚C.  Using the Fisher Scientific Sonic Dismembrator 550 (Pittsburgh, PA), samples were sonicated with a microtip on ice for sixteen cycles of 10 sec on, and 60 sec off on the “microtip” setting to fragment the majority of DNA within 200-800bp range.  Sonicated extract containing 2mg of protein was immunoprecipitated as previously described (Zhong et al., 2010) with 7.5µg of anti-GFP antibody (gift from Anthony Hyman) or anti-Pol III antibody (gift from Jason Lieb). ChIP-seq library preparation for enriched DNA and the genomic DNA input control for at least two biological replicates was performed as described in (Zhong et al. 2010), except Qiagen MinElute PCR purification and gel extraction kits were used after the Klenow step.  Libraries were multiplexed as described in Lefrancois et al. (2009) in order to sequence four samples in a single flow cell using the Illumina Genome Analyzer II platform. L1 animals were fed bacteria expressing RNAi clones from the Ahringer library (Fraser et al., 2000) until collection at the young adult stage. RNAi-treated young adults were harvested in TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was then treated with DNA-free DNaseI (Ambion, Austin, TX). A total of 5 µg of DNase-treated RNA harvested from snpc-4(RNAi) and wildtype young adult animals was subjected to the Illumina small RNA v1.5 sample preparation kit and 22-30 bp small RNAs were sequenced
attributes.tag: GEO Accession
attributes.value: GSM1291079