RNA Isolation and Transcriptome Analysis of Nodulating Roots

Total RNA was isolated from the nodulating and non-nodulating root tissue by using LiCl precipitation method and a quality check was carried out on the bioanalyser as described by Pradhan et al. [25 (link)]. mRNA was purified from total RNA samples using PolyATtract mRNA Isolation System according to the manufacturer’s instructions (Promega Corporation, Madison, WI, USA). Four cDNA libraries (3 libraries of nodulating tissue at various stages and one control) were generated using the Universal RiboClone cDNA Synthesis System (Promega Corporation, Madison, WI, USA). Double-stranded cDNA was synthesized using the Universal RiboClone cDNA Synthesis System (Promega Corporation, Madison, WI, USA) using random hexameric primers and following the manufacturer’s protocol. All cDNA libraries were sequenced using the Roche GS FLX Titanium series sequencing reagents and sequencer. A stringent quality filtering using the NGS ToolKit [33 (link)] was done with average quality score of all the reads above 30, with cut off phred quality score of 20 for over 70% of individual read length. Reads were aligned to genome of M. ciceri [34 (link)] (ASM18590v1) using BLASTN with e-value 1E-05, and reads mapped to M. ciceri genome were discarded.