My one c1 streptavidin dynabeads

For each family of mature tRNAs (with introns removed and containing terminal CCA) with a similar consensus sequence and a common anticodon, a pair of probes is designed so that, upon annealing to the complementary tRNA, the resulting nick in the DNA-RNA hybrid is located at the site of the anticodon. We designed a total of 67 probe-pairs to cover the majority of cytosolic tRNAs. The downstream probes were 5′-phosphorylated to enable enzymatic ligation. Small-RNAs extracted from cells were subjected to deacylation and biotinylation, followed by hybridization with the probe library. The DNA/RNA hybrids were then subjected to overnight ligation with T4 DNA ligase. MyOne-C1 Streptavidin Dynabeads (Invitrogen) were then used to purify biotinylated DNA/RNA hybrids, and the ligated probes were then eluted after incubation with RNase H and RNase A followed by incubation with an elution buffer (50 mM Tris [pH 8], 10 mM EDTA, 1% SDS; incubate at 65°C for 30 min with intermittent vortexing). Eluted probes were subsequently cleaned up and minimally PCR amplified (12–15 cycles) for high-throughput sequencing.

tRNA profiling was conducted as previously described^{8 (link)}. Briefly, samples were deacylated with 0.1 M Tris-HCl (pH 9) at 37°C for 30 minutes, precipitated, and biotinylated using Pierce RNA 3’ End Biotinylation Kit (Thermo Fisher), with 0.66 pmol of yeast tRNA-Phe added as a spike-in standard. Following chloroform extraction, biotinylated RNA was hybridized to DNA probe pairs complementary to the 3’ and 5’ arms of each tRNA isoacceptor. Nicks at the anticodon loop of DNA-RNA hybrids were ligated using SplintR ligase and T4 DNA ligase (NEB). DNA-RNA hybrids were purified using My-One-C1 Streptavidin Dynabeads (Invitrogen), and ligated probes were eluted after Rnase H and Rnase A treatment. Probes were PCR amplified and sequenced using Illumina NextSeq (MidOutput, 150 SR) at the Rockefeller University Genomics Center. For computational analysis, fastq files were aligned to tRNA probe sequences using bowtie2, and reads were further sorted, indexed, and counts were generated with samtools. Raw counts were imported into R 4.0.2 and differentially expressed transcripts determined using DESeq2.