title: Long-read nanopore PCR-cDNA sequencing of pea stipules
design: Long-read nanopore PCR-cDNA sequencing of pea stipules
library_construction_protocol: Whole stipules were harvested from the third leaflet node of individual seedlings 2 weeks post-emergence and snap-frozen in liquid nitrogen. Each sample was crushed into a fine powder by pestle and mortar. Samples were then weighed on a microgram scale to 1 mg of whole stipule tissue per extraction.  P. sativum cultivars 'Thomas Laxton' and 'Daisy' (supplied by the Australian Grains Genebank and the John Innes Centre) were grown within a controlled glasshouse environment at an average ambient temperature of 22°C and subjected to long-day light cycles (8 h dark, 16 h light). The soil medium macro/micro nutrients and other constituents consisted of 0.5 L Vermiculite, 0.5 L Perlite, 35 g Macracote Coloniser Plus 4-month slow release fertiliser (15N : 3P : 9K), 30 g Nitrogen slow-release fertiliser (40N : 0P : 0K), 25 g Water holding granules, 15 g Trace elements (6Mg : 6.5FE :5.4S : 1.5Mn : 0.4Zn : 0.14B : 0.07Mo), and 5 g Garden lime per 30 L of soil. Total RNA was then isolated using Invitrogen TRIzol reagent (ThermoFisher Scientific) using the manufacturer's protocol. Minor modifications to the protocol included an extended centrifugation for 15 minutes, as some RNA precipitate was still suspended in solution for some samples. RNA precipitate pellets were washed twice rather than once, as it was found this removed further impurities and improved the overall quality of the RNA. Centrifugation between washes was also extended to 10 minutes, to ensure the RNA pellet was anchored to the bottom of the Eppendorf tube during wash steps. Total RNA pellets for each sample were then re-suspended in RNase-free water, and subsequently quality-checked and quantified utilising the NanoDropTM spectrophotometer.  A PCR-cDNA Nanopore dataset with 12 samples (3 replicates across 4 conditions) from pea (Pisum sativum) species was prepared with the Oxford Nanopore PCR-cDNA Barcoding kit (SQK-PCB109) following the manufacturer's guidelines. Fifty-nanograms total RNA input was used for each of the 12 samples. Samples were reverse-transcribed into cDNA and PCR amplified and barcoded for 15 cycles in a thermocycler.