title: GSM4453643: F1_pGC_replicate2; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: Total RNA of animals was isolated by TRIzol extraction ~4 hours after RNA injection. RNAs ranging from approximately 15 to 30 nucleotides were gel-purified from total RNA (20 ug) on a 15% polyacrylamide/urea gel. Size-selected small RNAs species were ligated to a 3' adapter (/5rApp/CTGTAGGCACCATCAAT/3ddC/) using T4 RNA ligase 2, truncated (NEB). Samples were treated with Antarctic Phosphatase (NEB) followed by treatment with T4 polynucleotide kinase (NEB). 3'-ligated small RNAs were hybridized to (5' GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG), a primer that will eventually be used for reverse transcription. 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 small RNAs using T4 RNA ligase I (NEB). After reverse transcription, libraries were amplified and multiplexed with a 6-nucleotide 3' barcode, before pooling for next-generation sequencing on a NextSeq500.
attributes.tag: GEO Accession
attributes.value: GSM4453643