GSM5025697: Total_small_RNA_dis3(ust56); Caenorhabditis elegans; miRNA-Seq
(1) Total RNAs were isolated from L3 stage worm using a dounce homogenizer (pestle B) in TRIzol solution (Invitrogen) followed by DNase I digestion (Fermentas, no. 18068015). 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. (2) 3xFLAG::GFP::NRDE-3-associated siRNAs were isolated from L3 stage worm lysates. The worm lysates were generated via Bioruptor UCD-200 (Diagenode). The lysate was pre-cleared with protein G-agarose beads (Roche) and incubated with anti-FLAG M2 agarose beads (Sigma #A2220). The beads were washed extensively and were eluted with 100 μg/ml 3xFLAG peptide (Sigma). The eluates were incubated with TRIzol reagent followed by isopropanol precipitation and DNase I digestion (Fermentas). To facilitate 5′-phosphate-independent deep sequencing, the precipitated RNAs 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 Novogene (Beijing, China). sRNAs ranging from 18 to 40 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.