RNA-seq of Vigna vexillata (A408): root: waterlogged
Oneg of total RNAs was used to generate a sequencing library using an NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturers instructions. The mRNA fragmentation and priming was performed using NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified (by AxyPrep Mag PCR Clean-up (Axygen)) double-stranded cDNA was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing.