Illumina NovaSeq 6000 paired end sequencing; Molecular origins of short-term genome instability following chromosome mis-segregation
Molecular origins of short-term genome instability following chromosome mis-segregation
For Reversine/DMSO treatment: hTERT RPE-1 cells were synchronized with a single thymidine block. After thymidine release, synchronized cells were treated with DMSO or 500 nM Reversine for 24 hours. After Reversine/DMSO washout, cells were allowed to divide for about 60 hours. Then, they were either harvested or incubated with the fluorescent substrate of the B-galactosidase enzyme DDAO-Galactoside (DDAOG) 10 mM for 90 minutes. At the end of the incubation, cells were harvested for FACS sorting and acquired using a FACSAria Fusion flow cytometer (BD). Cells were gated for singlets and alive cells and then FSC-A, SSC-A and Alexa-647 intensity were used to distinguish cycling from arrested cells, i.e. cycling cells were gated using the same Alexa-647 Mean Fluorescence Intensity of the control sample without DDAOG, while arrested cells were gated imposing 0,1% on Alexa-647 signal to the control sample without DDAOG. For whole-transcriptome analysis, RNA was isolated using the RNeasy mini kit (Qiagen) in accordance to manufacturer's protocol and RNA quality was examined using the Agilent 2100 Bioanalyzer (Agilent Technologies). 500 ng of total RNA was used as input for library preparation using TruSeq total stranded RNA Illumina.