title: GSM9409521: Resistant rapeseed treated by submergence-bio rep 2; Brassica napus; RNA-Seq
library_construction_protocol: Total RNA of rapeseed seedling sample was extracted by using Trizol reagent. RNA quality check, mRNA extraction, cDNA reverse transcription, and preparation of amplicon library were performed by Biomark Biotechnology Co., Ltd (Beijing, China). Purity, concentration and integrity of RNA sample were examined by NanoDrop, Qubit 2.0, Agilent 2100, etc. Qualified RNA were processed for library construction. The procedures include: (1) mRNA was isolated by Oligo(dT)-attached magnetic beads. (2) mRNA was then randomly fragmented in fragmentation buffer. (3) First-strand cDNA was synthesized with fragmented mRNA as template and random hexamers as primers, followed by second-strand synthesis with addition of PCR buffer, dNTPs, RNase H and DNA polymerase I. Purification of cDNA was processed with AMPure XP beads. (4) Double-strand cDNA was subjected to end repair. Adenosine was added to the end and ligated to adapters. AMPure XP beads were applied here to select fragments within size range of 300-400 bp. (5) cDNA library was produced by certain rounds of PCR on cDNA fragments. To ensure the quality of library, Qubit 2.0 and Agilent 2100 were used to examine the concentration of cDNA and insert size. Q-PCR was processed to obtain a more accurate library concentration. Library with concentration larger than 2 nM is acceptable. RNA-seq was conducted on an Illumina NovaSeq6000 platform (Illumina Inc., San Diego, USA) following the standard Illumina PE150 protocol.