miRNA-Seq analysis of parental human leukemia K562 and clonal etoposide resistant K562 (designated K/VP.5) cell lines.

We report that ~300 mature miRNAs were detected in these cells. 73 miRNAs were overexpressed (fold change > 2; adjusted P-value < 0.05) and 14 were underexpressed (fold change > 2; adjusted P-value < 0.05) in K/VP.5 compared to K562 cells. Overall design: A mirVana miRNA Isolation Kit with phenol was used to isolate total RNA from K562 and K/VP.5 cells. The total RNA was subsequently DNase I treated and concentrated on a spin-column. NEB Next Small RNA libraries were prepared form the purified K562 and K/VP.5 total RNA samples and the libraries were size selected using Sage Pippin (130-200 bp range, adapters are ~128 bp) with an average final library size of 180 bp and sequenced on MiniSeq as 2 x 75 bp reads. 3' adaptor sequences and low quality bases were trimmed from 75 base pair paired-end sequence reads using Cutadapt v2.4 (Martin, 2011). Trimmed reads with a sequence length between 15 and 50 base pairs were aligned to potential contaminant sequences of human genome (ribosomal RNAs and tRNAs) using Bowtie v1.1.2 (Langmead et al., 2009) with a seed length of 18 and allowing zero mismatches in the seed. Then, remaining reads were aligned to miRbase (Griffiths-Jones, 2004) v21 mature sequences using Bowtie v1.1.2 with a seed length of 18, allowing 1 mismatch in seed and reporting maximum 5 alignments per read. Finally, remaining reads were aligned to human reference genome, GRCh38, using Bowtie v1.1.2 with a seed length of 18, allowing 1 mismatch in seed and reporting only unique alignments for each read. Using Samtools (Li et al., 2009) and in-house R scripts, total number of reads for each mature miRNA was counted.