title: PromethION cDNA sequencing of GM12878
design: *RNA isolation and cDNA preparation*We isolated total poly-A RNA from flash-frozen GM12878 cell pellets usingthe protocol that was previously used by the Nanopore RNA consortium (https://protocolexchange.researchsquare.com/article/a97320b0-e61e-45b6-a744-bd5b658cf91f/v1).We performed reverse transcription, strand switching, cDNA synthesis, andPCR amplification following the consortium protocols as well. The PCRreaction involved 11 cycles using the standard ONT PCR primers provided intheir SQK-LSK308 kit.*PromethION sequencing and basecalling*We adapted the cDNA  products for nanopore sequencing using the standardlibrary preparation protocol for the ONT SQK-LSK109 sequencing kit. Eachlibrary was then loaded on a PromethION flow cell and sequenced using MinKNOWsoftware and run scripts provided by ONT. Nanopore data were basecalledusing Guppy version 3.0.5 and the flip flop model.*Sequence alignment*Nanopore-derived sequences were aligned to GRCh38 using minimap2 (v 2.1) usingsplice aware setting (-ax splice -uf -k14). The resulting sam file wasconverted to a sorted bam file using samtools (v1.3.1).