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. 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.

本研究通过RNA测序（RNA-seq），探究靶向目标小开放阅读框（sORFs）/微蛋白的单导向RNA（sgRNA）处理所诱导的基因表达变化。为验证此类转录变化是否可在微蛋白拯救-绿色荧光蛋白（GFP）融合细胞中被逆转，本研究对拯救细胞与亲代细胞的转录组差异进行了比较。本次RNA测序在源自A375的细胞系中开展了三次生物学重复。所有细胞系均携带组成型表达的Cas9-T2A-BSD-TagBFP转基因（Addgene #196714），且拯救细胞额外包含带有GFP标签的密码子修饰微蛋白转基因。在RNA提取前，A375 Cas9对照细胞系与微蛋白拯救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。