RNA Extraction, Library Preparation, and Illumina MiSeq Sequencing

Leaf samples selected for NGS analysis were dried using a clean, absorbent paper towel to remove the RNAlater^® and then powdered in liquid nitrogen with sterile mortars and pestles. Total RNA was extracted from 0.1 g of leaf samples using the RNeasy^® plant mini Kit (Qiagen Inc.), following the manufacturer’s instructions. The RNA was eluted in 70 μl of RNase-free water, visualized on a 0.8% (w/v) agarose gel electrophoresis at 100 V for 30 min, and its quantity was measured using the ssRNA assay on the Qubit^® 2.0 fluorometer (Invitrogen^TM) system. The extracted RNA was then stored at −80°C. The cDNA libraries were prepared from 1 μg of the total RNA using the Illumina TruSeq^® RNA sample preparation protocol according to the manufacturer’s instructions (Illumina, San Diego, CA, United States). Briefly, poly-A containing mRNA molecules were purified using oligo-dT, and then fragmented into small pieces using the Illumina “Elute, Prime, Fragment Mix.” The fragmented RNA was copied into the first-strand cDNA using reverse transcriptase and random primers, and second-strand cDNA was synthesized using DNA polymerase I and RNase H. The double-stranded cDNA was purified using Agencourt AMPure^® XP magnetic beads (Beckman Coulter, Inc., Indianapolis, IN, United States). The end-repair of synthesized cDNA was performed using End Repair mix. Thereafter, 3′ ends were adenylated and unique adaptors for each library ligated to the 5′ and 3′ ends ds cDNA. The dsDNA was enriched through PCR to create the final cDNA library under the following cycling conditions: one cycle of 98°C for 30 s; 15 cycles of 98°C for 10 s; 60°C for 30 s; and 72°C for 30 s, with a final extension of 72°C for 5 min.

The final size and concentration of the cDNA libraries were estimated with the Agilent Tape Station 2200 system (Agilent Technologies, Santa Clara, CA, United States) and Qubit^® 2.0 fluorometer (Invitrogen^TM), respectively. The cDNA libraries, each with unique adaptor, were normalized to 4 nm and pooled for multiplex sequencing. A pooled library consisted of 24 biological samples, each at equal molar concentration (hence, two flow cells) were used. The libraries were sequenced using a 2 × 300 cycle PE V3 Illumina kit (Illumina, San Diego, CA, United States). Paired-end reads were generated using the Illumina MiSeq System at the BecA-ILRI Hub in Nairobi, Kenya.