GSM4629525: 28 day old lettuce leaves, replicate 3 [L101]; Lactuca sativa; RNA-Seq
Total RNA was extracted from each of the three lettuce leave replicates (denoted as L93, L96 and L101) with the RNeasy Plant Mini Kit following the manufacturer's protocol (Qiagen, Valencia, CA, USA). RNA was DNase-treated with the optional on column digestion following the manufacturer's protocol (Qiagen, Valencia, CA, USA).  A modified enrichment method was developed to process the RNA samples for compatibility with Nanopore library preparation protocols.35 Using an average of 14µg total RNA, the Invitrogen Dynabeads polyA enrichment protocol (Thermo Fisher, Carlsbad, CA, USA) was performed keeping the polyA depleted material and discarding the polyA enriched material. The polyA depleted material was then purified with the Zymo RNA clean and concentration kit according to the manufacturer's protocol (Zymo Research, Irvine, CA, USA). Purified RNA was processed using the Invitrogen RiboMinusTM plant kit for removal of plant ribosomal RNA per the manufacturer's protocol (Thermo Fisher, Carlsbad, CA, USA). 1-10ug of host depleted RNA form each sample was then A-tailed using Poly(A) polymerase (New England Biolabs, Ipswich, MA, USA) for 25 mins at 37ºC.  RNA quantity and quality were assessed with QubitTM fluorometer (Version 4.0, InvitrogenTM, Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) respectively. Bioanalyzer 6000 Nano RNA Eukaryotic program was used to assess the quality of the RNA. The samples were stored at -80°C until ready for library preparation. Library preparation for cDNA-PCR sequencing was performed using the SQK-PCS109 sequencing kit following the manufacturer's instructions (Oxford Nanopore Technologies, Oxford, UK). Briefly, 50ng of processed RNA sample from L93, L96 and L101were reverse transcribed using Maxima H Minus Reverse Transcriptase (Thermo Fisher), and left to incubate in the thermal cycler at 42°C for 90 min and 85°C for 5 min. To amplify the products without introducing biasness, full length transcripts were selected by PCR using LongAmp Taq 2x Master Mix (New England Biolabs, Ipswich, MA, USA) with the following cycling conditions, an initial denaturing step at 95°C for 5 min, then 14 cycles of 15 s at 95°C, 15 s at 62°C, and 1 min at 65°C, followed by a final extension step at 65°C for 6 mins and a hold at 4°C. The products were then using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN) with 1.8 (v/v) ratio of beads to reaction mixture. cDNA library using 100 fmol of amplified cDNA with adapters was loaded onto a FLO-MIN106 flowcell and the RNA library was sequenced on a MinION device for 48 h. Basecalling was perform in real-time using the MinIT device. L93, L96 and L101 replicates were sequenced on individual flowcells FAL87564, FAL08147, and FAL1190 respectively to provide information about the process reproducibility and variability.