title: GSM8332878: TG,Elevated CO2,July,R6; Glycine max; RNA-Seq
library_construction_protocol: Total RNA was extracted using the RNeasy Plant Mini Kit (74904; Qiagen, Germany) according to the manufacturer's protocol with the following modification: RPE buffer wash was increased from one to 4-5 times. Further, the total RNA sample was digested with RNase-Free DNase Set (79254; Qiagen, Germany) to remove any traces of genomic DNA contamination. Total RNA concentration was initially evaluated using NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Scientific™, USA). The quality of each RNA sample was determined in 1.2% agarose-TAE (0.5x) gel electrophoresis containing SYBR™ Safe DNA Gel Stain (Thermo Fisher, USA) by evaluating the integrity of the 28S and 18S ribosomal RNA bands and absence of smears. The total RNAswere converted into individually barcoded polyadenylated mRNAseq (PolyA) libraries with theKapa Hyper stranded mRNA Sample Prep kit (08098123702; Roche, IN, USA). Libraries were barcoded with Unique Dual Indexes (UDI's) which have been developed to prevent index switching.The adaptor-ligated double-stranded cDNAs were amplified by PCR for 8 cycles with the Kapa HiFi polymerase (07958927001; Roche, IN, USA). The final libraries were quantified with Qubit (ThermoFisher, MA, USA) and the average cDNA fragment sizes were determined on a Fragment Analyzer. The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Biorad, CA, USA) for accurate pooling of barcoded libraries and maximization of number of clusters in the flowcell.