The effect of CBX2 knockdown on gene expression in MDA-MB-231 cells

To identify potential CBX2-regulated genes in triple negative breast cancer cells we assessed global gene expression changes in CBX2-depleted MDA-MB-231 cells by RNA-sequencing analysis using the Illumina NovaSeq 6000 platform. Through Gene Set Enrichment Analysis we identified that CBX2 promotes cell growth, mTORC1 activation, E2F signalling and inhibits RBL2-mediated DREAM-complex activity. Overall design: MDA-MB-231 cells were transfected with either a non-silencing scrambled control siRNA (siSCR) or CBX2 targeting siRNAs (siCBX2#2/siCBX2#3), using Lipfectamine RNAiMAX (Invitrogen) to a final concentration of 25 nM. Cells were grown for 72 hours prior to RNA extraction using the RNeasy mini kit (Qiagen). Library preparation and sequencing using the Illumina NovaSeq 6000 platform was performed by Novogene Co Ltd (Cambridge, UK). Read alignment was performed using the HISAT2 algorithm and FPKM calculated to estimate gene expression in each sample.