Instrument: PromethION
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Frozen tissues kept at -80°C were homogenized with a mortar and pestle in liquid nitrogen. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) followed by a column clean-up using the Direct-zol RNA Mini Prep Plus kit (Zymo Research, Irvine, CA) and performing an in-column DNA digestion. Integrity of the DNase-treated RNA was verified on the Experion electrophoresis system (Bio-Rad, Hercules, CA). For each sample, 50 ng total RNA was transferred to 0.2 ml PCR tubes and adjusted to a final volume of 9µl with nuclease free water. Reactions were prepared (9 μl total RNA, 1 μl 10 µM VNP primer , 1 μl 10mM dNTPs) and incubated for 5 min at 65°C, then snap cooled on a pre-chilled freezer block. Strand-switching buffer (4 μl 5x RT buffer, 1 μl RNaseOUT, 1 μl nuclease-free water, and 2 μl 10 μM strand-switching primer) was then added to the snap-cooled, annealed mRNA, and incubated at 42°C for 2 min. One μl of Maxima H Minus Reverse Transcriptase was added, and reactions were incubated at 42°C for 90 min, 85°C for 5 min, and held at 4°C. A round of PCR was used to introduce barcodes to the cDNAs using Oxford Nanopore PCR barcoding expansion 1-96 kit (Cat. No.EXP-PBC096). Barcoding PCR reactions were set up for each cDNA (1 μl PCR barcode, 19 μl first-strand cDNA, 20μl LongAmp Taq 2x master mix), and cycled for [3 min at 95°C]x1, [15 sec at 95°C, 15 sec at 62°C, 7 min at 65°C] x13 cycles, [15 min at 65°C]x1, then held at 4°C. Each barcoded cDNA was purified in 1x Ampure XP Beads, eluted in 20µl of nuclease free water and quantified in the Qubit. Barcoded cDNAs were pooled in a final volume of 47µl and the pool was taken to the DNA Technologies Core and Expression Analysis Laboratory at the University of California Davis where they performed the adapter ligation on the cDNA pool using SQK-DCS109 kit following manufacturer's guidelines. 50 fmol of adapter ligated library was loaded onto PromethION flow cell vR9.4.1. Long-read Nanopore cDNA sequencing