title: GSM4038392: WT_3_R; Brassica rapa; RNA-Seq
library_construction_protocol: Tissue disruption, mRNA isolation, library synthesis, sequencing, and fastq processing were conducted by Amaryllis Nucleics (Oakland, CA). Leaf tissue was disrupted with Zirconia/Silica beads for 1 minute in a MiniBeadbeater-96 (BioSpec Products, Inc.) while frozen. After adding 500 µL of Lysis/Binding Buffer to each sample and vortexing until homogeneous, samples were run on the MiniBeadbeater-96 for an additional minute. Following tissue disruption samples were centrifuged at 16,000 x g for 10 minutes at 20 ºC. For each sample, a 50 µL aliquot of the supernatant was added to 50 µL of NEB RNA binding buffer and mRNA isolated as per the NEBNext® Poly(A) mRNA Magnetic Isolation Module manual. RNA-seq libraries were prepared by using the Full Transcript mode YourSeq Dual (FT & 3'-DGE) RNAseq Library Kit (Amaryllis Nucleics). A Bioanalyzer 2100 (Agilent, High Sensitivity DNA Kit) was used for library quality control, to determine average library size, and together with concentration data from a Qubit 2.0 Fluorometer (Life Technologies, dsDNA High Sensitivity Assay Kit) to determine individual library molarity and pooled library molarity. Pooled libraries were sequenced on a NextSeq 500 (Illumina, High Output v2 75 cycle kit) to yield single-read 80 bp reads.