GSM4718808: rRNA_COLD_CDE1_CDE1GFP; Caenorhabditis elegans; OTHER
total RNA were extracted from L3 larva, digested by DNase I, and then ligated to the following 3′ RNA linkers with T4 RNA ligase (Thermo #EL0021) (1 μg total RNA, 2 μl 3′ RNA linker (10 μM), 1 μl 10× T4 RNA ligation buffer, 2 μl T4 RNA ligase) by incubating at 37 °C for 40 min. PCR Free Briefly, total RNA were extracted from L3 larva, digested by DNase I, and then ligated to the following 3′ RNA linkers with T4 RNA ligase (Thermo #EL0021) (1 μg total RNA, 2 μl 3′ RNA linker (10 μM), 1 μl 10× T4 RNA ligation buffer, 2 μl T4 RNA ligase) by incubating at 37 °C for 30 min.  3' RNA linker-1:   5'-pGATCCACACTCGGGCACCAAGGATTTAACCGCGAATTCCAGC-NH2-3' (the underlined sequence served as a barcode for sample labeling).  3' RNA linker-2:   5'-pCGACACACTCGGGCACCAAGGATTTAACCGCGAATTCCAGC-NH2-3'  3' RNA linker-3:   5'-pGTACCACACTCGGGCACCAAGGATTTAACCGCGAATTCCAGC-NH2-3'.  The RNAs were reverse transcribed with the following primer:  Universal 3' linker RT:   5'-GCTGGAATTCGCGGTTAAATCCTTGGTGCCCGAGTGT-3'. The cDNAs were PCR amplified (16 cycles) with the primers 26S rRNA-F: 5'-CAGATCACTCTGGTTCAATGTC-3' and universal 3' linker RT primer, or with the primers 5.8S rRNA-F: 5'-GGTTGCATCGAGTATCGATGAA-3' and universal 3' linker RT primer, gel purified and then deep sequenced using an Illumina platform, according to the manufacturer′s instructions, by Novogene (Beijing, China). The number of reads with distinct 3'-end modifications were counted by custom Perl scripts. Three biological replicates were conducted in each genotype or treatment. PCR free
GEO
GSM4718808