title: RNA-seq of human breast cancer cell line MCF7 overexpressing different full length SOX2, N-terminal SOX2 and C-terminal SOX2 with nuclear export signal
design: RNA-seq of human breast cancer cell line MCF7 overexpressing different full length SOX2, N-terminal SOX2 and C-terminal SOX2 with nuclear export signal
library_construction_protocol: Cells were propagated to 70-90% confluence in T175cm2 flasks, incubated with cycloheximide (100 µg/ml, C7698, Sigma) 30min prior to harvest (scraping), washed (2x) with cold PBS buffer and resuspended in 400µl of 1x lysis buffer (#9803, CST) supplemented with protease/phosphatase inhibitor cocktail (#78442, Thermo Fisher) and 5mM of MgCl2 (HN03.1, ROTH). Cells were mechanically disrupted by vortexing (3x 10sec at max. output, 10min on ice in between), cleared by centrifugation (10min, 21.000rcf, 4°C) Hot phenol extraction of RNA TruSeq RNA Library Prep Kit v2 Illumina