RNA-seq of Glycine max: no treatment
RNA of pool replicates was extracted using the RNeasy mini total RNA isolation kit (Qiagen, Valencia, CA, USA). RNA integrity, purity, and concentrations were assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, Palo Alto, CA, USA).Messenger RNA (mRNA) was purified from the total RNA with oligo-dT beads provided in the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Beverly, MA, USA). Complementary DNA (cDNA) libraries for Illumina sequencing were constructed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB, Beverly, MA, USA) and NEBNext Multiplex Oligos for Illumina (NEB, Beverly, MA, USA). The mRNA was chemically fragmented and primed with random oligos for first-strand cDNA synthesis. The double-stranded cDNA was then purified, end repaired and a-tailed for adapter ligation. Following ligation, the samples were selected and sample-specific indexed. The final quantified libraries were pooled in equimolar amounts for sequencing on an Illumina HiSeq. 2500 utilizing a 125-bp read length with v4 sequencing chemistry (Illumina, San Diego, CA, USA).