NCLX KO
The mRNA was isolated (minimum 3 g) using the RNeasy Mini Kit. A fragmentation buffer (Ambion) was used to short fragment the mRNA. These short-fragmented mRNA were used as a template to synthesize double-stranded cDNA. The cDNA was end-repaired and ligated to Illumina adapters. Further, to generate the library, these cDNAs were size selected (~250 bp) on an agarose gel, and PCR amplified. To sequence the cDNA, the prepared cDNA library was fed to the Illumina HiSeq 2500 sequencing platform (Berry Genomics). The expression level of each transcript of a gene was quantified using read counts with HTseq.