Zr small rna page recovery kit

The procedure was adapted from a previous report (49 (link)). Briefly, RNA species smaller than 200 nt were extracted from the total RNA using RNA Clean & Concentrator Kits (Zymo Research). The tRNA fraction was further extracted from the small RNAs by using 6% TBE–urea gel and extracting the migrated tRNAs from the gel using a ZR small-RNA PAGE Recovery Kit (Zymo Research).

RNA-seq libraries were prepared from samples from three independent experiments. 10 µg of total RNA was run on 15% Urea-TBE gel, and 20–50 nt (tiRNA fraction) or 50–110 nt (tRNA fraction) was gel-purified using ZR small-RNA PAGE Recovery kit (Zymo Research). The purified RNAs were treated with calf intestinal alkaline phosphatase (New England Biolabs). After purification using Direct-zol RNA Microprep (Zymo Research), the RNAs were treated with T4 polynucleotide kinase (New England Biolabs), and then purified using Direct-zol RNA Microprep. Small RNA libraries were prepared using the TruSeq Small RNA library preparation kit (Illumina) according to the manufacturer’s protocol. Sequencing was performed on the Illumina platform (Molecular Biology Core Facility, Dana-Farber Cancer Institute, Boston, United States), and 75 bp single-end reads (tiRNA fraction) or 150 bp single-end reads (tRNA fraction) were generated.

5′ and 3′ RACEs were performed as described previously (57 (link)) with slight modifications. Briefly, So-RNaseZ-digested products of tRNA precursor transcripts were purified on 10% urea-PAGE gels using a ZR small-RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA), and then the 5′ and 3′ ends were ligated with 50 pmol of universal microRNA (miRNA)-cloning linker 5′-CAGACUGGAUCCGUCCUC-App-3′ and 5′-AppCUGUAGGCACCAUCAAU-ddC-3′ (synthesized by Integrated DNA, Coralville, IA, USA), respectively, with the addition of 20 U of T4 RNA ligase (Ambion, Austin, TX, USA) in the presence of 40 U of RNasin-Plus. The reaction mixtures were incubated at 16°C for 16 h, and the 5′- and 3′-linker-ligated RNAs were recovered by isopropanol precipitation and then mixed with 2 pmol of random primer and 100 pmol of 3′ RACE RT primer (5′-ATTGATGGTGCCTACAG-3′; complementary to the universal miRNA cloning linker), respectively. Full-length cDNAs were synthesized by using 200 U SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The 5′ and 3′ end-containing cDNA fragments were next amplified using nested PCR using primers listed in Table S1. PCR products were recovered from a 2% (wt/vol) agarose gel and then TA-cloned into pMD19-T (TaKaRa, Dalian, China), followed by colony PCR and sequencing.