Small RNA sequencing of Boerhaavia diffusa
In order to initialise the RNA extraction procedure, the tissues were gathered from RNAlater solution and fixed by using Liquid Nitrogen, followed by manual grinding of frozen tissues by using mortal and pestel to transform in a fine powdered form. The finely powdered samples precisely weighing 500 mg were subjected to RNA extraction at 65  for 30 min by using one mL of the CTAB buffer (Hexadecyltrimethylammonium bromide 2% W/V; PVP-40 2% W/V; 100 mM TrisHCl pH 8.0; 25 mM EDTA, 2 M NaCl; and 2% -mercaptoethanol fresh) for further lysis of the cells. Further RNA was precipitated at 4  overnight and Following the addition of an equal amount of chloroform:isoamylalcohol (24:1) over supernatant, 1/10th volume of lithium chloride (LiCl) was added, followed by collection of supernatant after 5 min of incubation. In due course 70% ethanol (EtOH) was used to rinse the obtained precipitated RNA followed by resuspension of RNA in 30 uls of RNase free Water. Further DNA removal was performed by using Turbo DNAse with manufacturers protocol. Ultimately, the quality of extracted RNA was assured by employing Nanodrop 1000 spectrometer as well as Agilent Tapestation was utilised for RIN value evaluation.