RNAseq profiling of eSCC human cell line (KYSE-410) knocked-out for SHIP2 using CRISPR/Cas9 technology

In our study, we used mRNA sequencing to investigate the effect of SHIP2 ablation on the transcriptome of KYSE-410 cells. Overall design: CRISPR/Cas9 technology was used for SHIP2 ablation in KYSE-410 cells using the CRISPR/Cas9n plasmids. Plasmids were co-transfected with a GFP-based CRISPR reporter (Addgene, #48138) using the Lipofectamine CRISPRMAX according to the manufacturer's instructions. GFP-positive cells were subjected to FACS sorting Fluorescence-activated cell sorting using a FACSAria III (BD Biosciences, San Jose, CA, USA) and collected in a 96-well plate. Cells were expanded and positive clones were screened by Western blot for SHIP2. Once expanded RNA from validated clones (D4, G4, G10 and H3) as well as control colony (transfected with the same vector devoid of gRNA) was extracted using the MicroElute Total RNA kit (Omega Biotek, R6831-02) according to the manufacturer's recommendations with DNase I digestion protocol on column.