Massively parallel splicing reporter assay on designed sequence libraries

We designed 4 oligonucleotide libraries containing either a retained intron, a cassette exon, tandem 5' or tandem 3' splice sites, cloned them into dedicated reporter constructs, transfected and integrated these constructs in the genome of K562 cells, and performed targeted RNA sequencing to determine RNA splicing ratios and a FACSseq approach to determine protein isoform ratios. Overall design: Massively parallel reporter assay for alternative splicing

本研究设计了4种寡核苷酸文库（oligonucleotide libraries），分别包含保留内含子（retained intron）、盒式外显子（cassette exon）、串联5'剪接位点或串联3'剪接位点；将上述文库克隆至专用报告基因构建体（reporter constructs）中，随后转染并整合至K562细胞的基因组内；通过靶向RNA测序（targeted RNA sequencing）测定RNA剪接比率，并采用荧光激活细胞分选测序（FACSseq）技术检测蛋白质异构体比率。整体实验设计：面向可变剪接（alternative splicing）的大规模平行报告基因检测实验（Massively parallel reporter assay）。