miRNA sequencing from Nicotiana rustica leaves
Small RNA sequencing (smRNA) libraries were constructed using QIAseq miRNA Library Kit protocol (Qiagen, Maryland, U.S.A.) following manufacturers instructions. Briefly, 63ng 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 Unique Molecular Index (UMI) assignment by priming with reverse transcription primers. cDNA thus formed was enriched and barcoded by PCR amplification (17-19 cycles). The Illumina-compatible sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA). The fragment size distribution of the library was analyzed on Agilent 2200 TapeStation.