GSM4627048: smallRNA_N2_48h_rep3; Caenorhabditis elegans; ncRNA-Seq
Worms were collected in TriReagent (Thermo Fisher Scientific) and underwent three freeze-thaw cycles. Following addition of 1-bromo-3-chloropropane (BCP), the aqueous phase was then precipitated with isopropanol at -80°C for 2 hrs. To pellet RNA, samples were spun at 21,000 x g for 30 min at 4°C. After three washes in 75% ethanol, the pellet was resuspended in water. RNA concentration and integrity was measured using a TapeStation (Agilent Technologies). 16-30 nt small RNAs were size-selected from 5 ug total RNA on 17% denaturing polyacrylamide gels. Small RNAs were treated with 5' polyphosphatase (Illumina) to reduce 5' triphosphate groups to monophosphates to enable 5' adapter ligation. Small RNA-sequencing libraries were prepared using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina (NEB). Small RNA amplicons were size-selected on 10% polyacrylamide gels and quantified using qRT-PCR. Samples for each developmental time point were pooled into a single flow cell and single-end, 75 nt-long reads were generated on a Illumina NextSeq 500 (Illumina).
GEO Accession
GSM4627048