RNA-seq of Homo sapiens peripheral blood lymphocytes
The SQK-PCS108 cDNA-PCR Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK) was used according to the manufacturer's instructions. Briefly, mRNA was denatured at 65C for 5 minutes. SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) and buffer were used in reverse transcription, which included a strand-switching component to create double-stranded cDNA. A short PCR was performed to add on barcodes and rapid-attachment primers. Rapid-sequencing adapters were added with a short incubation at room temperature. Samples were quantified with the QuBit dsDNA HS kit before loading on MinION. Sequencing was performed on MinION (Oxford Nanopore Technologies), using standard flow cells FLO-MIN106D R9.4.1 and MinKNOW software version 3.1.13. Samples were sequenced for a mean duration of 18 hours (range, 15.5 to 22 hours). Fast5 files from the MinION software were used for downstream analysis. Base-calling and demultiplexing were performed on all samples, except cDNA014, with Albacore software version 2.3.4 (Oxford Nanopore Technologies). Because of the timing of the experiments, Albacore was no longer available for cDNA014; thus it was base-called and demultiplexed with Guppy software version 2.3.1 (Oxford Nanopore Technologies). Fast5 files were processed for quality and binned into folders indicating pass or fail. The passed files were base-called, and if a sample was barcoded, it was demultiplexed according to the barcodes, and an output fastq file was created. Adapter trimming was performed using Porechop software version 0.2.3 (https://github.com/rrwick/Porechop, last accessed January 5, 2019), with sequences of middle adapters discarded.