alias: GSM1919774
title: GSM1919774: hrde-1_23C_G1_rep2_sRNAseq; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: Total small RNA was extracted from frozen synchronized adult worm grinds with the mirVana microRNA (miRNA) isolation kit (Ambion). small RNA was first ligated to the IDT linker-1 oligo (5' rAppCTGTAGGCACCATCAAT/3ddC/ 3') using T4 RNA ligase 2, truncated  from NEB, and then treated with antarctic phosphatase (NEB) to remove the mono- or tri-phosphate at the 5' end of the small RNAs. Antartic phosphatase was heat-inactivated and T4 polynucleotide kinase reaction  (with ATP) was used to add 5-mono-phosphate group to the small RNA. 33-54 nt small RNA-linker-1 ligation product was gel purified using 8% PAGE with 7.5M urea. A linker oligo with 4 nt barcode (/5AmMC6/ ACG CTC TTC CGA TCT r#r#r#r#-3') was ligated to the 5' end of the small RNA-linker-1 ligation product. cDNA was synthesized with primer (5'-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGCTGGCTACAG-3') and SuperScript® III Reverse Transcriptase from Invitrogen. cDNA was PCR amplified with primers:5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-CAAGCAGAAGACGGCATACGAGAT ###### GTGACTGGAGTTCAGACGTGTGCTCTTCC -3' ( ######: 6 nt multiplexing index sequence). Small RNA library was then purfiled using 3% agarose gel.
identifiers.namespace: GEO
identifiers.id: GSM1919774