RNA Extraction and Sequencing Protocol

For RNA extraction, cells were pelleted, frozen in liquid nitrogen, and stored at −80°C until RNA extraction. RNA extraction using Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Maryland, USA, Cat# 80204) per manufacturer’s instruction with cell homogenization in RLT buffer via QIAshredder (Qiagen, Maryland, USA, Cat# 79656). RNA quality control using Agilent Tapestation 2200 (Agilent, California, USA, Cat# G2964AA) and the Agilent RNA ScreenTape (Agilent, California, USA, Cat# 5067- 5576) and Agilent RNA ScreenTape Sample Buffer and Ladder (Agilent, California, USA, Cat# 5067- 5577, Cat# 5067- 5578) per manufacturer’s instruction. Library prep using Illumina RNA library prep kit (Illumina, California, USA, Cat # RS-122-2001) and sequenced on an Illumina HiSeq 4000 paired end using manufacturer’s instructions.
Sequencing reads aligned to Hg38 using HISAT2 (version 2.0.5), assembly and quantification was performed using StringTie (version 1.3.3) and differential expression was performed using R package Ballgown (version 2.6.0) as described (Pertea et al., 2015 (link), 2016 (link)). Exon skipping was determined using IGV Viewer Sashimi Plots (Robinson et al., 2011 (link)).