miRNA sequencing of MCF-10A
Small RNA sequencing (smRNA) libraries were prepared with NEXTflex Small RNA sample preparation protocol (Bioo Scientfic Corporation, Austin, Texas, U.S.A.). 1000 ng of Qubit quantified total RNA was used as starting material. 3 adapters were ligated to the specific 3OH group of micro RNAs followed by ligation of 5 adapter. Adapter ligated fragment was reverse transcribed with M-MuLV Reverse transcriptase by priming with reverse transcription primers. cDNA thus formed was enriched and barcoded by PCR amplification. Library was size selected using Polyacrylamide gel electrophoresis. Illumina-compatible sequencing library was quantified and its fragment size distribution was analyzed on Agilent 2100 Bioanalyzer. Small RNA sequencing (smRNA) libraries were prepared with miRNA Library Kit as per manufacturer protocol. The Illumina-compatible sequencing libraries were quantified and fragment size distribution of the libraries was analyzed on Agilent 2200 TapeStation. The sequencing was carried out using 75 cycles SE chemistry on Illumina NextSeq 550 High Output platform following manufacturers procedure.