Soybean (Glycine Max) zone of nodulation transcriptome in response to inoculation with Nod factor deficient Bradyrhizobium japonicum (NodC-) Lane2/2
Total RNA was extracted from roots of six to eight plants inoculated with either WT or nodC− mutant B. japonicum at 48 h p.i. These samples were subsequently used for library generation.  The mRNA-seq libraries were constructed following Illumina’s standard protocol. Briefly, mRNA was isolated from total RNA using Sera-Mag Magnetic Oligo(dT) Beads. Isolated mRNA was fragmented using divalent cations under increased temperatures followed by ethanol precipitation. The fragmented mRNA was reverse-transcribed into cDNA using Superscript III and random primers. The cDNA was end-repaired to create blunt-ended fragments, and an ‘A’ base was ligated to the 3′ ends of the fragments to create an ‘A’ base overhang. Adapters with a ‘T’ base overhang were ligated to each end of the cDNA fragments. The ligated fragments were run on an agarose gel, and a thin slice of DNA fragments corresponding to approximately 200 bp was excised and purified from the gel. The purified size-selected fragments were enriched by PCR to generate the final library fragments.  To differentiate the reads that arise from one gene to those from the duplicated gene, sequencing was run with a relatively long read length of 75 bp. Furthermore, each library was run on two lanes for greater sensitivity.