| Full name | Hydrolysis-Based tRNA Sequencing |
| Summary | hydro-tRNAseq is based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing |
| Key findings | By combining with SSB PAR-CLIP, Hydro-tRNAseq maps pre-tRNA transcription initiation and termination boundaries |
| Sequencing strategy | RNA-Seq |
| Raw data | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE95683 |
| Experimental protocol | Total RNA from HEK293 (Flp-In T-REx, Invitrogen) was isolated using TRIzol (Invitrogen). For each sample, 20 μg total RNA was resolved on 12% urea-polyacrylamide gels and recovered within a size window of 60–100 nt. The eluted fraction was subjected to limited alkaline hydrolysis in a 15-μL buffer of 10 mM Na2CO3 and 10 mM NaHCO3 (pH 10.3) at 60°C for either 10 min (replicate 1) or 1 hr (replicates 2–4). The partially hydrolyzed RNA was dephosphorylated with 10 U calf intestinal phosphatase (New England Biolabs) in a 50-μL reaction of 100 mM NaCl, 50 mM Tris-HCl (pH 7.9) at 25°C, and 10 mM MgCl2, 1 mM DTT, 3 mM Na2CO3, and 3 mM NaHCO3, at 37°C for 1 hr. The resulting RNA was re-phosphorylated with 10 U T4 polynucleotide kinase (NEB) in a 20-μL reaction of 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM DTT, and 1 mM ATP at 37°C for 1 hr. Fragments of 19–35 nt were converted into barcoded small RNA cDNA libraries, and sequenced on an Illumina HiSeq 2500 instrument. |
| Analysis protocol | Adapters were trimmed using cutadapt. Sequencing read alignments were performed using the Burrows-Wheeler aligner against an in-house curated and annotated list of mature and pre-tRNAs containing predicted tRNA sequences for human genome version hg19 (http://gtrnadb.ucsc.edu). Sequencing reads were first mapped against mature tRNAs. Remaining reads were mapped against genomic tRNA sequences that included 5′ leader and 3′ trailer sequences, as well as tRNA introns. |
| Organism/cell line | Homo sapiens (HEK293) |
| Conditions | alaline hydrolysis time (10' vs 1h) |
| Approximate experimental time | 1-2 days |
| Starting RNA amount | 20 µg total RNA |
| tRNA expression | + |
| Base modifications | + |
| Charging status | - |
| tRNA processing and fragmentation | + |
| Citation | https://doi.org/10.1016/j.celrep.2017.07.029 |
