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.