The cDNA reads of A549 treated with IFN beta generated by Nanopore GridION long-read sequencer
Cell were seeded in T25 flasks overnight. The next day media was replaced with either media containing 2 ng/mL IFN (treated) or normal media without IFN (mock). Total RNA was prepared from cells 24 hrs post-treatment using the RNeasy Mini Kit (Qiagen). PolyA+ RNA was enriched from total RNA using the Dynabeads mRNA Purification Kit (Invitrogen). cDNA libraries were prepared from 200 ng of PolyA+ RNA using the Direct cDNA Sequencing Kit (Oxford Nanopore), according to the PCR-free 1D read protocol for full-length cDNA (Oxford Nanopore, SQK-DCS109), with some modifications. Specifically, RNase Cocktail Enzyme Mix (ThermoFisher) was used during the RNA digestion step after the first-strand synthesis; all reaction amounts for reverse transcription reactions up to the second-strand synthesis step were doubled; from second-strand synthesis up to adapter ligation, reactions were 1.5x of original amounts; during adapter ligation, 35 ul Blunt/TA Ligase Master Mix was used instead of the recommended 50 ul, and nuclease-free water was excluded. Final libraries were loaded into MinION Fluidics Module flow cells (Oxford Nanopore, FLO-MIN106D), and sequencing was carried out on GridION MK1 and MinION MK1C instruments (Oxford Nanopore) for 3 days, using default parameters. The FASTQ files generated by Nanopore GridION long-read sequencer were trimmed using Porechop (https://github.com/rrwick/Porechop) and aligned to the hg19 genome using Minimap2 (https://github.com/lh3/minimap2) with -ax splice command. The output SAM files were then converted to indexed, sorted BAM files using SAM tools (https://github.com/samtools/). The BAM files were visualized with the UCSC genome browser.