Peanut plant metatranscriptome analysis

Plant RNA was isolated from the peanut roots using the Trizol® reagent (Invitrogen, Carlsbad, USA) method, following the manufacturer’s instructions. The average sample RNA integrity number (RIN) was 8.1, as determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (Supplementary Fig. S1). Poly(A) mRNA was then separated from the total RNA using oligo(dT) magnetic beads (Invitrogen) and fragmented into ca. 200-bp pieces using a fragmentation solution (Ambion, USA). These mRNA fragments were used as templates in a random hexamer-primed cDNA synthesis reaction performed using reverse transcriptase (Invitrogen). Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA synthesis kit (Invitrogen). cDNA was then purified using the QIAquick PCR extraction kit (Qiagen, Germany) and, following end-repair and poly(A)-processing, ligated with sequencing adaptors. The libraries were prepared for sequencing on an Illumina HiSeq 4000 platform (Illumina, USA), following manufacturer’s protocols.