T4 rna ligase

Three replicates of CV-A10-infected cells at different time were mixed together and used for RNA extraction. Briefly, total RNA from the harvested cells was extracted with the Trizol reagent (Invitrogen, USA). The concentrations of total RNA of samples were determined by NanoDrop 2000, and the RNA integrity and purity was assessed using an Agilent Bioanalyzer 2100. All RNA integrity numbers (RINs) for the samples were > 7.0 and rRNA 28S/18S ≥ 1.6, indicating that the RNA was of good quality and suitable for constructing small RNA libraries.
Subsequently, the qualified RNA samples were fractionated by 15% denaturing polyacrylamide gel electrophoresis (PAGE), and then small RNA fragments between 18 and 35 nt in length were isolated from the gel. The sRNA molecules were ligated to a 5’ adaptor and a 3’ adaptor by T4 RNA ligase (Promega, USA). Next, the adapter-ligated sRNAs were converted to cDNA by RT-PCR following the Solexa sequencing protocol (Illumina, San Diego, CA, USA). Small RNA sequencing was performed in ANOROAD Genome Inc. (Beijing, China) using a NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) on the Illumina HiSeq™ 2500 platform.

The genome segments of WALV, MUDV and WARV were reverse-transcribed using a ‘full-length amplification of cDNA' (FLAC) technique described by Maan et al [37] (link). Briefly, a 35 base self-priming oligonucleotide ‘anchor-primer’ with a phosphorylated 5′ terminus was ligated to the 3′ ends of the viral dsRNAs using the T4 RNA ligase, followed by reverse transcription using RT system (Promega). The resulting cDNAs were amplified using complementary primers to the anchor primer. The resulting cDNA amplicons were analyzed by agarose gel electrophoresis. For cloning purposes, a high fidelity KOD polymerase enzyme (Novagen) was used in the PCR.

The 5′ and 3′ ends of tRNA^Met and tRNA^Gln from the control cell line C19 and mutant cell line III-3 were sequenced after cDNA synthesis, PCR amplification, and cloning, as detailed elsewhere (38 (link)). First, total mitochondrial tRNA was circularized by incubation in the presence of T4 RNA ligase (Promega) to ligate the 3′ and 5′ ends of tRNAs. Then, complementary DNA chains of tRNA^Met and tRNA^Gln were synthesized using reverse transcriptase after annealing the circular tRNA to the specific oligodeoxynucleotides MET1 (5′-TATGGGCCCGATAGCTTATTTAGCT-3′) and GLN1 (5′CAAAATTCTCCGTGCCACCTATCA-3′), respectively. The second strands of these cDNAs were then synthesized by using primers MET2 (5′-CCCCGAAAATGTTGGTTATACCCTT-3′), GLN2 (5′-GATTCTCAGGGATGGGTTCGATT-3′), respectively. The artificial tDNAs were then amplified by PCR, using above primers, respectively. Those resultant PCR products were cloned in the TA vector (Invitrogen), and eight clones of each control cell tDNA and III-3-1′s tDNA were analyzed by Sanger sequence.

The cleavage sites of predicted targets were validated through 5’RLM-RACE. Briefly, the isolation of mRNA/Poly A⁺ RNA from total RNA was performed using PolyATtract mRNA isolation system IV (Promega, Madison, WI, U.S.A.) and 25 ng of mRNA was ligated to the RNA adapter using T4 RNA ligase (Promega). Further, amplification of cleaved products of miRNA target genes was performed using reverse primers specific to target gene and primers complementary to RNA adapter. The PCR amplified products were purified by gel elution, cloned into a pGEMT Easy vector (Promega) and sequenced. The sequencing results were analysed to map the cleavage sites. Primers used in 5’ RLM-RACE are detailed in the S1 Table.