RNA seq of lettuce: Red light
Library preparation for sequencing was performed using the TruSeq RNA Sample Prep Kit v2 (Illumina). A total of 200 ng of total RNA was purified by oligo-dT beads, then poly (A)-containing mRNA were fragmented into small pieces with Elute, Prime, Fragment mix. First-strand cDNA was generated by First Strand Master Mix and Super Script II (Invitrogen) reverse transcription kit under the following conditions: 25 C for 10 min, 42 C for 50 min, 70 C for 15 min, followed by addition of second strand master mix, and synthesis of second strand at 16 C for 1 hour. The purified fragmented cDNA was combined with End Repair Mix, incubated at 30 C for 30 min, and the end-repaired DNA was purified with the Ampure XP Beads (AGENCOURT). Further, A-Tailing Mix was added, mixed well by pipetting and incubated at 37 C for 30 min, followed by the addition of RNA index adapter and incubation at 30 C for 10 min. The end-repaired cDNA was purified with Ampure XP Beads (AGENCOURT). Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments. Then the PCR products were purified with Ampure XP Beads (AGENCOURT). The libraries were amplified on cBot to generate the cluster on the flowcell (TruSeq PE Cluster Kit V3cBotHSIllumina). The amplified flowcell were then sequenced to obtain paired-end reads using the Illumina HiSeq 2000 sequencer.