title: STIM1/2 DKO_34
design: To perform RNA-seq mRNA was isolated from total RNA (minimum 3 g) from HCT116, STIM1 KO, STIM2 KO, and DKO HCT116 cells using the RNeasy Mini Kit. Short fragment off mRNA was generated using fragmentation buffer (Ambion). The double-stranded cDNA was generated, end-repaired, and ligated to Illumina adapters. These cDNAs were size selected (~250 bp) and PCR amplified. The cDNA library was fed to the Illumina HiSeq 2500 sequencing platform (Berry Genomics) and the expression level of each transcript of a gene was quantified using read counts with HTseq. The genes with  identifiers that has zero read counts were removed from each sample, and the biomaRt R package (Durinck et al., 2009) was used to convert Ensemble gene identifiers to gene symbols using the January, 2019 archive (http://jan2019.archive.ensembl.org). Identifiers corresponding to multiple gene symbols were removed, and based on thresholds defined the edgeR R package (McCarthy et al., 2012; Robinson et al., 2010) was used to identify lowly expressed genes.