Microprotein knockout in rescue cell lines and control conditions. Microprotein knockout in rescue cell lines and control conditions

RNA-seq sequencing was carried out to investigate which expression alterations were induced by treatment with sgRNAs targeting the sORFs/microproteins of interest. To test whether any of these changes could be reversed in the microprotein rescue-GFP fusion cells, transcriptional alterations of rescue and parental cells were compared. Overall design: RNA-Seq was performed in triplicates in cell lines derived from A375. All cell lines carried a constitutively expressed Cas9-T2A-BSD-TagBFP transgene (addgene #196714) and rescue cells additionally contained a GFP-tagged codon-altered microprotein transgene. A375 Cas9 control and microprotein rescue cell lines were treated with either sgRNA targeting the respective microprotein or targeting a control locus (TRAC) before RNA extraction.

本研究借助RNA测序（RNA-seq），旨在探究靶向目标小开放阅读框（small open reading frames, sORFs）/微蛋白的单向导RNA（single guide RNA, sgRNA）处理所诱导的基因表达改变。为验证此类表达变化是否可在微蛋白救援-绿色荧光蛋白（green fluorescent protein, GFP）融合细胞中得到逆转，本研究对救援细胞与亲本细胞的转录组差异进行了比较。
整体实验设计：在源自A375细胞株的多个细胞系中开展三次生物学重复的RNA测序。所有细胞系均携带组成型表达的Cas9-T2A-BSD-TagBFP转基因（Addgene #196714），其中救援细胞系额外带有经密码子修饰的GFP标记微蛋白转基因。在进行RNA提取前，A375 Cas9对照细胞系与微蛋白救援细胞系分别接受靶向对应微蛋白的sgRNA，或靶向对照位点TRAC的sgRNA处理。