Breast Cancer miRNA Sequencing Protocol

Small RNA sequencing was performed on single lane of Illumina HiSeq 1000 with eight multiplex libraries from the four breast cancer cell lines. The reads obtained from deep sequencing of small RNAs were subjected to Illumina adaptor trimming using FastX tool kit and were size filtered to select for candidate miRNA's (14 to 24 bases) from a pool of small RNA sequences using in-house perl script. The size separated reads were then mapped onto human miRNA reads obtained from miRBase (version 21) using Bowtie2 (version 2.1.0)¹⁶ (link) with 0 mismatches in the first 8 bases. MicroRNAs were quantified followed by normalisation by read per million using in-house script. Deregulated miRNAs with > = 3 fold change were retained for further analysis. For searching microRNAs targeting PR 3′UTR, differentially expressed microRNAs in response to progesterone were compared to microRNAs predicted to target PR using 6 algorithms (TargetScan, miRanda, miRWalk, miRMap, RNA22 and RNAhybrid).