rRNA Depletion and RNA-seq Library Preparation

NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #Z1955E) was chosen to remove the targeted ribosomal RNA (rRNA). All RNA with a percentage of RNA fragments > 200 nucleotides (DV200) ≤ 50% skipped fragmentation and proceeded to library preparation. After rRNA depletion and fragmentation, cDNA synthesis and NGS library preparation were performed using NEBNext^® Ultra™ II Directional RNA Library Prep Kit (NEB#E7760L). The library was quantitated using Qubit 3.0 (life Invitrogen, USA) and quality was assessed with LabChip GX Touch (PerkinElmer, USA). After removal of terminal adaptor sequences and low-quality data by using fastp (version: 0.19.5) [15 (link)] and removal rRNA reads through aligning clean reads to rRNA database (download from NCBI) by using bowtie2 (version:2.2.8) [16 (link)], clean reads without known rRNA were aligned to the reference human genome (hg19) through STAR (version 020201) [17 (link)]. Fusions were detected by a customized version of Arriba 1.1.0. and annotated by in house software annoFilterArriba (version:1.0.0) with NCBI release 104 database. All final candidate fusions were manually verified with the integrative genomics viewer browser. A series of quality control metrics was computed by using RNA-SeQC assessment [18 (link)]. A threshold of ≥ 80 million mapped reads and ≥ 10 million junction reads per sample was set.