title: GSM2417590: total NRDE-3 bound small RNA in eri-1 after cold shock; Caenorhabditis elegans; miRNA-Seq
library_construction_protocol: NRDE-3-associated siRNAs were isolated from embryo lysates as described (Zhou, X et al, Genetics 197, 121-32 (2014)). Briefly, the animals were sonicated in lysis buffer (20 mM Tris-HCl [pH 7.5], 200 mM NaCl, 2.5 mM MgCl2, and 0.5% NP-40); the lysate was pre-cleared with protein G-agarose beads (Roche), and incubated with anti-FLAG M2 agarose (Sigma) beads. The beads were washed extensively, and FLAG::GFP::NRDE-3 and associated RNA were eluted with 100 μg/ml 3xFLAG peptide (Sigma). The eluates were incubated with TRIzol reagent (Invitrogen), followed by isopropanol precipitation. The precipitates were treated with calf intestinal alkaline phosphatase (CIAP, Invitrogen), re-extracted with TRIzol, and treated with T4 polynucleotide kinase (T4 PNK, New England Biolabs) in the presence of 1 mM ATP. Small RNAs were cloned and deep sequenced using an Illumina platform, according to the manufacturer's instructions, by the Beijing Genomics Institute (BGI Shenzhen). sRNAs ranging from 20 to 30 nt were gel-purified and ligated to a 3’ adaptor (5’-pUCGUAUGCCGUCUUCUGCUUGidT-3’; p, phosphate; idT, inverted deoxythymidine) and a 5’ adaptor (5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’). The ligation products were gel-purified, reverse transcribed, and amplified using Illumina’s sRNA primer set (5’-CAAGCAGAAGACG GCATACGA-3’; 5’-AATGATACGGCGACCACCGA-3’). The samples were sequenced using an Illumina Hiseq platform.
attributes.tag: GEO Accession
attributes.value: GSM2417590