Characterizing BLV miRNA Expression Using RNA-seq

HEK293T cells (6-well format) were transfected with 2 µg of BLV miRNA expression vectors using Lipofectamine 2000. RNA was extracted 48 h post transfection and fractionated on a 15% PAGE-urea gel. Small RNAs (<70 nt) were isolated as previously described (31 (link)) and treated with or without RNA 5′ polyphosphatase. RNA was then recovered via ammonium acetate precipitation. 5′-end characterization as described above, was performed to confirm miRNA expression and dephosphorylation by the RNA 5′ polyphosphatase treatment. Small RNA libraries were then prepared (GSAF, UT Austin, USA) for Illumina small RNA sequencing (RNA-seq) using the multiplex small RNA library prep set (New England Bio Labs) and sequenced on a Illumina HiSeq 2500. Adapter sequences were trimmed from the reads using custom Python scripts. The preprocessed reads were then mapped to the respective pri-miRNA sequences using the SHRiMP2 software package (33 (link)). To analyze the fold change of the 5′-start position of putative B5 pre-miRNAs with RNA 5′ polyphosphatase, we counted RNA-seq reads of RNAs approximately pre-miRNA length (>50 nt), in which the 5′-nucleotide perfectly mapped to the BLV B5 miRNA locus (30 nt upstream and 54 nt downstream of the 5′-end of BLV-miR-B5 5p).