RNA sequencing of fixed or live FACS-sorted GLF16+ and GLF16- cells

Li-Fraumeni-p21WAF1/Cip1 Tet-ON cells were seeded onto 10-cm cell culture plates and once they reached 70% confluency they were treated with 10 microgram/ml Doxycycline for 6-8 days so that cells become senescent. Untreated cells were also cultured to serve as negative and unstained controls during the sorting analysis. For sorting of fixed cells, cells were subsequently collected, fixed with PFA (4%, 15 min), permeabilized with 90% methanol and stained with GLF16 (10 microgram/ml, 8 min). For live cell sorting, doxycycline-treated cells were inoculated with mGLF16 for 3h and subsequently washed twice with PBS. In both cases, GLF16+ cells were sorted against GLF16- using FACSAria II and collected in 5ml sterile, RNAse-free tubes. Total mRNA from sorted cells was extracted using the NucleoSpin(R) totalRNA FFPE kit (Macherey-Nagel, Germany) (in the case of fixed cells) or the NucleoSpin RNA mini kit (Macherey-Nagel, Germany) in the case of live cells. cDNA libraries were prepared using the NEBNext ultra II stranded RNASeq kit (Reverse strand specificity) and products were single-end sequenced at 101 bp length using the HiSeq4000 platform (Illumina)). Each experiment was performed twice.