GSM3560719: 89-gem-Day3-OEA085; Caenorhabditis elegans; RNA-Seq
Samples of at least 1000 worms were washed 3X in M9 by settling then snap-frozen in Trizol and kept at -80C until library-prep. Samples were freeze-thawed 10 times to break worm cuticle and RNA was precipitated using chloroform-isopropanol. RNA was treated with 10U DNase I (Roche #04716728001) for 20 minutes at 37°C and purified on Qiagen RNeasy columns (QIAGEN 15596-026). Libraries were prepared using combined protocols from SuperScript II Reverse Transcriptase (Invitrogen 18064-014), NEBNext mRNA Second Strand Synthesis Module (E6111), and NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB-E7645). Half reactions were used for all steps except the final PCR amplification. Briefly, Oligo d(T)25 beads (NEB-S1419S) were washed and resuspended in 2X RNA Binding Buffer (2 M LiCl , 80 mM Tris HCl (pH 7.5), 4 mM EDTA , 0.2% Triton X-100) in a 1:1 ratio and 500ng of total RNA was added. RNA was denatured by incubating at 65C for 5 minutes and mRNA was allowed to bind to beads. Beads were washed twice in Wash Buffer (150 mM LiCl , 20 mM Tris HCl (pH 7.5), 1.0 mM EDTA , 0.01% Triton X-100) and mRNA eluted in 10 mM Tris Buffer by incubating at 80C for 2 minutes. An equal volume of Binding Buffer was added, washed once more, and the mRNA was eluted into a First Strand Synthesis Reaction Buffer containing 4μl 5X First-Strand Buffer (Invitrogen), 1μl 50µM Random hexamers (Invitrogen N8080127), 0.5μl 10mM dNTP mix, and 1.5μl 0.1M DTT. 0.5μl RNaseOUT (Invitrogen 10777-019), 2.5μl 0.1μg/μl Actinomycin D (Sigma A1410), and 0.5μl SuperScript II Reverse Transcriptase were added and samples were incubated in a thermal cycler for 10 minutes at 25°C, 15 minutes at 42°C, and 15 minutes at 70°C. Second strand synthesis was performed immediately using the NEBNext mRNA Second Strand Synthesis Module. Libraries were then generated using the NEBNext Ultra II DNA Library Prep Kit. For PCR library enrichment, 13 cycles of amplification were performed. Library quality was assessed on a Bioanalyzer High Sensitivity DNA Chip (Agilent 5067-4626) and concentration was determined using KAPA Library Quantification Kit (KK4824). Libraries were sequenced on an Illumina HiSeq 2500 system by the Babraham Sequencing Facility.
GEO Accession
GSM3560719