title: GSM2694129: N2_PolyA-2; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: (RNAseq) Worms were broken by bead beating (Minibead Beater; Biospec Products) in homogenization buffer [10 mM KCl/1.5/1 mM DTT/10 mM Tris·HCl (pH 8.0)/50 mM Sucrose/0.05% Nonidet P-40/1 mM complete protease inhibitors (Sigma #P2714) at a 1:1 ratio of 0.1mm glass beads to packed worm volume for 3 cycles of 30 secs at 5,000 rpm. Extracts were used immediately for immunoprecipitations and equal concentration of extract as determined by protein quantification was added to each IP (ChIPseq) All worms were maintained and grown on op50 seeded NGM plates and harvested in water. The worms were washed three times in water, and the bacteria were cleared by sucrose flotation. The worms were then frozen in liquid nitrogen and ground to a powder with a mortar and pestle. The resulting worm powder was transferred into cross-linking buffer (1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM EDTA, 1 mM EGTA, 1% formaldehyde, phosphate-buffered saline [PBS]) for 10 min at room temperature. The reaction was quenched for 5 min at room temperature by the addition of glycine to the mixture to a final concentration of 125 mM and sedimented at 4,000 × g. Pellets were washed three times with FA buffer plus 0.1% SDS (50 mM HEPES [pH 7.5], 1 mM EDTA, 1% Triton, 0.1% deoxycholic acid, 150 mM NaCl, 0.1% SDS) containing protease inhibitors. Each wash mixture was incubated for 5 min at 4°C and then sedimented at 4,000 × g. The pellets were then divided into 500-μl aliquots and stored at −80°C. Each aliquot was resuspended in 1.5 ml of FA buffer plus 0.3% SDS containing protease (Complete cocktail tablets, catalog number 11697498001; Roche) and phosphatase inhibitors (catalog number 786-450; GBiosciences). The samples were sonicated with a Virsonic digital 600 sonicator using a microtip (20 pulses of 11 s each at 30% amplitude with bursts of 0.9 s on and 0.5 s off) to generate DNA fragments of approximately 500 bp, as determined experimentally. Samples were sedimented at 13,000 × g for 15 min at 4°C, and the supernatant was transferred into a new tube, which was then diluted to 4.5 ml with FA buffer containing protein and phosphatase inhibitors. Total RNA sequencing libraries was extracted from whole animals by TRIzol extraction. Genomic DNA was removed using TURBO DNase (Invitrogen). Poly-A selected, single-ended, 125 bp strand-specific libraries were prepared by the UCCC Genomics Core (Aurora, CO) using the TruSeq Stranded mRNA Library Prep Kit (Illumina). For the J2-IP analyses, RNA recovered by immunoprecipitation with the J2 antibody (three biologically independent lysates) of young adult worms as well as input material (as a loading control) was converted into strand‐specific total RNA libraries using V2 Scriptseq (Epicenter #SSV21106) kits following manufacturer's instructions, except reverse transcription was done with SuperScript III (Invitrogen #18080‐044) using incrementally increasing temperatures from 42 to 59°C to allow for transcription though structured RNAs. rRNA was not removed from J2‐IP RNA samples. Immunoprecipitated DNA from ChIP samples were converted into sequencing libraries using ChIP‐Seq DNA Sample Prep Kit (IP‐102– 1001). Cluster generation and sequencing were performed on the Illumina HiSeq 2500 platform. The reads were de-multiplexed and converted to FASTQ format using CASAVA software from Illumina.
attributes.tag: GEO Accession
attributes.value: GSM2694129