MiRNA isoforms, tRNA-derived fragments, and rRNA-derived fragments have enrichments that differ characteristically among sub-cellular compartments and among cell lines

BT-20 (ATCC #HTB-19), MDA-MB231 (ATCC #HTB-26), and MDA-MB468 (ATCC #HTB-132) cells were grown according to standard cell growth conditions using Complete EMEM (Eagle's Minimum Essential Medium Medium) for BT-20 cells and DMEM (Gibco Dulbecco's Modified Eagle Medium) for MDA-MB-231 and MDA-MB-468 cells. Cell lines were tested frequently to ensure that they were mycoplasma-free. Cells were grown in 20-40 10 cm dishes depending on cell type until cells reached 90% confluence, then cells were washed two times with ice-cold 1X PBS. 2 ml of 1X ice-cold PBS were added to each plate and then mechanically scrape cells using a cell scraper. Cells were pooled into two 50 ml ice-cold conical tubes that we spun at 500 g for 10 minutes. Downstream sub-cellular fractionation followed by deep sequencing of total RNA, nuclear RNA (Nuc), cytoplasmic RNA (Cyto), mitochondrial RNA (Mito), and mitoplast RNA (MP) was done using our method Fraction-seq (see publication). Fraction-seq was done using biological replicates for each cell line: BT-20 (Rep1, Rep2, Rep3), MDA-MD-231 (Rep1, Rep2, Rep3), MDA-MB-468 (Rep1, Rep2, Rep3, Rep4) RNA libraries were prepared for sequencing using standard NEBNext protocols. The NEBNext 3'-adapter is AGATCGGAAGAGCACACGTCT. Short RNA-seq was run on the Illumina NextSeq 500 sequencer. A total of 50 samples were prepared for this study.