GSM9386557: ctrli_rep2; Caenorhabditis elegans; OTHER
Total RNAs were extracted from synchronized L4 stage animals by using TRIzol Reagent (Life Technologies, California, USA). The purified total RNAs were subjected to dephosphorylation by alkaline phosphatase (New England Biolabs, Ipswich, USA), phosphorylation by polynucleotide kinase (New England Biolabs, Ipswich, USA), and polyadenylation by E. coli Poly(A) Polymerase (New England Biolabs, Ipswich, USA), sequentially. Polyadenylated RNAs were purified by using DynabeadTM Oligo(dT)25 (Invitrogen, Waltham, MA, USA) and ligated to an RNA oligonucleotide adaptor by using T4 RNA ligase (New England Biolabs, Ipswich, USA). The ligated products were purified with DynabeadsTM Oligo(dT)25 (Thermo Scientific, Waltham, MA, USA) and used as a template in an RT reaction for the synthesis of first-strand cDNAs. The cDNAs were then amplified with SolgTM pfu DNA polymerase (Solgent, Daejeon, South Korea). The PCR products were cleaved with MmeI (New England Biolabs, Ipswich, USA), and the cleaved products with the length of 42 nucleotides (nt) were excised from 12% Tris-Borate-EDTA (TBE) gel. The fragments were ligated to a double-stranded DNA adaptor by using T4 DNA ligase (F. Hoffmann–La Roche, Basel, Switzerland). The ligated products (63 nt) were isolated by using gel extraction and PCR amplified with P5 primer and P7 index primer. Synchronized L4-prefertile wild-type worms under control or rnk-1 rnk-2 RNAi conditons were maintained on OP50 at 20°C, and harvested after washing three times independently using M9 buffer.For rnk-1 rnk-2 double RNAi experiments, the two double-stranded RNA (dsRNA)-expressing bacteria were separately cultured using liquid LB media containing 100 μg/mL ampicillin (USB, Santa Clara, CA, USA) at 37°C overnight until the optical density (OD) value at 600 nm reached 0.9. The bacteria were then mixed to 1:1 ratio. Total RNA was isolated using RNAiso plus (Takara, Japan) in accordance with the manufacturer's instructions. The PCR products (126 nt in length) were purified and subjected to high-throughput sequencing on Illumina HiSeq 2500 platform (Macrogen, Seoul, South Korea). Adaptors were removed from sequencing reads by using Trim Galore (v.0.6.6) (https://www.bioinformatics.babraham.ac.uk /projects/trim_galore).
GEO
GSM9386557_r1