Transcriptomic Analysis of Galectin-3 Knockdown

HeLa cells were transfected with siRNA to silence galectin-3 and with the non-silencing control siRNA against firefly luciferase. Total RNA was isolated with the RNeasy Mini Kit (Qiagen) and processed by the TruSeq® Stranded mRNA LT Kit to prepare RNA-seq libraries. The libraries were sequenced on an Illumina HiSeq 1500 sequencer via paired-end sequencing to obtain 2×50 bp paired reads. The obtained reads were mapped against the Homo sapiens genome reference (Ensemble Revision 74, hg19) using the STAR algorithm [23 (link)]. FPKM values were calculated for each sample, differential gene expression was analyzed using DEseq2 [24 (link)]. The genes with a FPKM value above 0.3 in at least one sample and a DEseq p-value of 0.05 or better were considered as differentially expressed if the absolute of the log2 fold change was one or larger. Differential exon usage was analyzed using DEXseq [25 (link)]. All algorithms used standard parametrization.