GSM2417599: 26S rRNA tail seq from eri-1 after cold shock; Caenorhabditis elegans; ncRNA-Seq
Total RNAs were isolated from bleached embryos. Briefly, embryos were harvested and dounced on ice with pestle B in TRIzol reagent (Invitrogen) followed by isopropanol precipitation. The RNA were digested with DNase I and re-purified by TRIzol reagent. Total RNAs were ligated to the 3' RNA linkers with T4 RNA ligase (Thermo #EL0021) (300 ng total RNAs, 2μl 3' RNA linker (10 ng/μl), 1μl 10x T4 RNA ligation buffer, 2μl T4 RNA ligase, 37°C, 30min ). 3' RNA linker-1: 5'-pGATCCACACTCGGGCACCAAGGATTTAACCGCGAATTCCAGC-NH2-3'. 3' RNA linker-2: 5'-pGTACCACACTCGGGCACCAAGGATTTAACCGCGAATTCCAGC-NH2-3'. 3' RNA linker-3: 5'-pCGACACACTCGGGCACCAAGG-NH2-3'. The RNAs were reverse transcribed with the following primers: 3' linker RT-1: 5'-GCTGGAATTCGCGGTTAAATCCTTGGTGCCCGAGTGTGGATC-3'. 3' linker RT-2: 5'-GCTGGAATTCGCGGTTAAATCCTTGGTGCCCGAGTGTGGTAC-3'. 3' linker RT-3: 5'-GCTGGAATTCGCGGTTAAATCCTTGGTGCCCGAGTGTGTCG-3'. The cDNAs were PCR amplified with the primers 26S rRNA-202-F: 5'-CATCTCTATCTCGTGGCAACACGAG-3' and linker-R: 5'-GCTGGAATTCGCGGTTAAATCCTTG-3' and gel purified. 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.
GEO Accession
GSM2417599