Instrument: MinION
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: RNA was extracted from naïve and activated CD8 T cells using the RNeasy Micro Kit (cat# 74004, Qiagen). ONT cDNA-PCR libraries were generated essentially according to the CELLO-seq protocol (Berrens et al, 2021, Nat. Biotech.; doi:10.1038/s41587-021-01093-1) with the following modifications: The addition of UMIs by splint ligation was omitted. Reverse transcription was performed using 1 ng total RNA isolated from a pool of cells. PCR reactions were performed using standard Oxford Nanopore Technologies barcoded primers under the following conditions: 90 °C for 45 s, 20 cycles (90 °C for 15 s, 64 °C for 30 s, 72 °C for 10 min), 72 °C for 5 min. PCR products were purified using 0.6X AMPure XP beads (Beckman). Equal quantities of each cDNA sequencing library were pooled using a total of 200 fmol, before sequencing on a MinION R9.4.1 flow cell using the SQK-LSK109 kit (1D sequencing). The standard MinKNOW procedure was used for a 72 hour run, according to manufacturers' instructions.