GSM4567617: N2_NoHS_rep3; Caenorhabditis elegans; RNA-Seq
Worms were lysed using a motor and pedestal, and RNA was extracted using Qiagen's RNeasy kit. RNA integrity was assessed using RNA 6000 Pico Kit for Bioanalyzer (Agilent Technologies #5067-1513) and mRNA was isolated using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). RNA-seq libraries were constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) following manufacturer's instructions. Briefly, mRNA isolated from ~200 ng - 1 μg of total RNA was fragmented to the average size of 200 nt by incubating at 94 °C for 15 min in first strand buffer, cDNA was synthesized using random primers and ProtoScript II Reverse Transcriptase followed by second strand synthesis using NEB Second Strand Synthesis Enzyme Mix. Resulting DNA fragments were end-repaired, dA tailed and ligated to NEBNext hairpin adaptors (NEB #E7335). After ligation, adaptors were converted to the 'Y' shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter #A63880) to generate fragment sizes between 250 and 350 bp. Adaptor-ligated DNA was PCR amplified followed by AMPure XP bead clean up. Libraries were quantified with Qubit dsDNA HS Kit (ThermoFisher Scientific #Q32854) and the size distribution was confirmed with High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies #5067- 4626). Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt to the depth of 20 million reads per sample following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4.
GEO Accession
GSM4567617