GSM3963335: WT_F5_R2; Caenorhabditis elegans; ncRNA-Seq
Adult worms were flash-frozen in liquid nitrogen. Total RNA was isolated by TRIzol extraction. RNAs ranging from approximately 15 to 30 nucleotides were gel-purified from total RNA (20 ug) on a 15% polyacrylamide/urea gel. Small RNA species were ligated to a 3' adapter (/5rApp/CTGTAGGCACCATCAAT/3ddC/) using T4 RNA ligase 2, truncated (NEB). To enable the cloning of 5'-triphosphorylated RNAs, samples were treated with Antarctic Phosphatase (NEB) followed by treatment with T4 polynucleotide kinase (NEB). Prior to ligation of the 5' adapter, 3'-ligated small RNAs and any excess 3' adapter were hybridized to the oligo that would eventually be used as a primer for reverse transcription (5' GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG). To help avoid cross-contamination, the 5' adapter was modified to contain the Illumina genomic sequencing primer annealing site followed by an additional 4 nucleotides at the 3' end. Two different 5' adapters (/5AmMC6/ACGCTCTTCCGATCTrArGrCrG and /5AmMC6/ACGCTCTTCCGATCTrCrGrUrC) were mixed in a 1:1 ratio, and the mix was ligated to each sample using T4 RNA ligase I (NEB). Every sample was treated with the same mix of 5' adapters. Libraries were amplified and multiplexed with a 6-nucleotide 3' barcode, then pooled for next-generation sequencing on a NextSeq500.