tRF and tiRNA Sequencing Protocol

tRNA-derived fragment and tiRNA sequencing was performed as previously described (25 (link)). Briefly, total RNA was extracted using TRIzol (Invitrogen) from three pairs of HASMCs with or without PDGF-BB (50 ng/ml) treatment for 24 h and quantified by a NanoDrop (Thermo Fisher Scientific, USA). tRNA-derived fragments (tRF and tiRNA) are heavily modified by RNA modifications that interfere with small RNA-seq library construction. RNA modifications need to be removed before library construction, which is the biggest difference from common small RNA sequencing. RNA modifications were removed by the rtStar™ tRF and tiRNA Pretreatment Kit (Arraystar, Rockville, MD, USA). A sequencing library specific for tRFs was constructed from the pretreated total RNA by the following steps: (1) 3′ and 5′-adapter ligations and m1A and m3C demethylation; (2) cDNA synthesis; (3) PCR amplification; and (4) to differentiate tRFs with full-length tRNA, sequencing libraries are size-selected for the RNA biotypes to be sequenced using an automated gel cutter. Size of 134–160 bp PCR-amplified fragments (corresponding to a 14–40 nt small RNA size range) were selected, while the full length tRNA is 76–90 nt. The libraries were denatured into single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as sequencing clusters and sequenced for 50 cycles on an Illumina NextSeq 500 system (Illumina, CA, USA) using a NeXTs 500/550 V2 kit (#FC-404-2005, Illumina, CA, USA). The sequencing data has been uploaded to the GEO database (GEO accession number GSE212737).
The abundance of tRF and tiRNA was evaluated using their sequencing counts and was normalized as counts per million of total aligned reads (CPM). Differentially expressed tRFs and tiRNAs analyses was performed with R package edgeR (33 (link)). Fold change (cut-off 1.5), p-value (cut-off 0.05) were used for screening differentially expressed tRFs and tiRNAs. The number of subtypes tRFs, which the CPM of the sample or the average CPM of the group is not less than 20, can be counted against tRNA isodecoders which share the same anticodon but have differences in their body sequence. The stacked bar chart is plotted with R barplot package.