GSM5572861: HT29_selumetinib_CCNB1_-ve_1; Homo sapiens; RNA-Seq
Cells grown in monolayers were harvested by trypsinization, centrifuged (300 x g for 5 minutes) and washed once with PBS prior to lysis in TRI reagent. For glyoxal fixation and staining, cell pellets were resuspended in 100 µL glyoxal fixation solution (2.8 ml water, 0.79 ml 100% ethanol, 0.31 ml 40% glyoxal and 30 µl acetic acid, pH 4-5) supplemented with 1:25 RNasin Plus RNase inhibitor (Promega, N261B) and incubated for 15 minutes on ice. The cell suspension was diluted in 1 mL PBS supplemented with 1:100 RNasin Plus RNase inhibitor and centrifuged at 2000  x g for 3 minutes at 4°C; the supernatant was discarded. All further centrifugation steps were carried out at similar conditions. Cells were permeabilized by adding 100 µL ice-cold methanol (100%, v/v) with 1:25 RNasin Plus RNase inhibitor drop by drop while gently vortexing cells, and incubated on ice for 30 minutes. The cell suspension was then diluted in 1 mL 1% bovine serum albumin (BSA) (Sigma, A7906) in PBS and centrifuged. For CCNB1 labelling, cells were incubated in 100 uL primary antibody (in this case CST 12231S - 1:200) diluted in 1% BSA in PBS with 1:25 RNasin Plus RNase inhibitor for 1 hour on ice. Following incubation, cells were washed once in 1mL 1% BSA in PBS. Secondary antibody staining was carried out in 100 μL Alexa Fluor 488 donkey anti-rabbit antibody (ThermoFisher Scientific, A21206) diluted 1:1000 in 1% BSA in PBS with 1:25 RNasin Plus RNase inhibitor and incubated on ice for 30 minutes in dark. Cells were washed once in 1 mL 1% BSA in PBS and the cell pellet resuspended in 200 µL 1% BSA in PBS with 1:100 RNasin Plus RNase inhibitor. Cells were incubated in 1 µg/mL DAPI. For collection of glyoxal-fixed cells that were stained with CCNB1 and flow sorted, cell suspension was pelleted by centrifugation at 2000 x g for 3 minutes at 4°C. Cell pellets were lysed in 1 mL TRI reagent (Sigma, T9424) and mixed until a homogeneous lysate was obtained. For phase separation, 0.2 mL chloroform was added, mixed thoroughly and allowed to stand for 10 minutes prior to centrifugation at 12,000 × g for 15 min at 4 °C. The upper aqueous phase containing RNA was transferred to a fresh tube, and 0.5 mL 2-propanol per mL original lysate was then added, samples mixed and allowed to stand for 10 minutes. Samples were centrifuged (12,000 × g for 10 min at 4 °C), and RNA pellets washed with 1 mL 75% (v/v) ethanol (7,500 × g for 5 min at 4 °C). Pellets were air-dried for 5-10 minutes and resuspended in RNase-free water. RNA prepared from flow sorted cells was quality controlled on a Bioanalyzer 2100 (Agilent) using RNA Pico 6000 chips (Agilent 5067-1513). mRNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760S) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490S), following the protocol provided by the manufacturer with the exception that two successive 0.9x AMPure purifications were performed on the final amplified libraries. All libraries were amplified using 12-14 PCR cycles. Library quality was assessed on a Bioanalyzer 2100 using High Sensitivity DNA chips (Agilent 5067-4626) and quantification performed with a KAPA Library Quantification Kit (Roche, KK4844). Libraries were sequenced by the Babraham Institute Next Generation Sequencing Facility on an Illumina HiSeq2500 in Rapid Run 50bp Single End mode.