RNAseq-PACS1-humanFibroblast
RNA sequencing (RNA-seq) was performed on total RNA extracted from immortalized human fibroblasts derived from PACS1 syndrome patients and healthy controls. Cells were either untreated or treated for 48 hours with 30 nM of either scramble siRNA or a mutation-specific siRNA targeting the PACS1 variant. RNA was extracted using silica column-based purification and treated with DNase to eliminate genomic DNA. RNA quality was assessed using the Agilent TapeStation 2200, with all samples showing RNA Integrity Number (RIN) values greater than 9.2. Mutation-specific PACS1 silencing was validated by RT-PCR prior to sequencing. Libraries were prepared using the Illumina Stranded mRNA Prep kit with poly(A) selection and sequenced on an Illumina NextSeq 2000 system, generating at least 2 × 30 million paired-end 100 bp reads per sample. Sequencing was performed at the Genotyping/Sequencing Platform.