Burro1 Nanopore sequencing and 5RACE
Nanopore sequencing libraries were prepared using the cDNA-PCR Sequencing Kit (SQK-PCS109, ONT) according to the manufacturers instructions. Additional RACE libraries were generated by reverse transcription and template-switching, using six reverse primers specific to Burro1. Nanopore amplicons were generated from cDNA with LongAmp Taq master mix (New England Biolabs, USA) and the Nanopore cDNA sequencing kit according to the manufacturers instructions. PCR products were purified using the AMPure XP beads (Beckman Coulter, USA). Sequencing was performed on a MinION Mk1B system (Oxford Nanopore Technologies Ltd., ONT), using a FLOMIN106 flow cell. Basecalling was performed using guppy (ONT guppy v4.4.1.). Adapters were trimmed using Porechop. Only reads with a minimum Q score of 10 were selected for analysis.