title: Time-series RNA-seq reads of SK-MEL-28 cells treated with two concentrations of vemurafenib
design: Time-series RNA-seq reads of SK-MEL-28 cells treated with two concentrations of vemurafenib
library_name: Sample 35_p
library_construction_protocol: The human melanoma SK-MEL-28 cell line was obtained from the ATCC (The American Type Culture Collection, Manassas, VA, USA). Cells were cultured in flasks in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 humidified incubator without antibiotics. For experimental studies, cells were maintained in the log phase of growth and treated at 80% confluency with 50 µM VEM (Vemurafenib, S1267, Selleckchem, Houston, TX, USA)(VEM 50) for 24 h or 10 µM VEM (VEM 10) for 8 days. After drug removal completely replacing the medium, cells were maintained by replenishing 30 % conditioned culture medium on days 5, 7, 9, 12, 15, 19, 22 for 50 µM VEM treatment. Using  10 µM VEM treatment, the medium was replaced with fresh medium (with added 10 µM VEM) on days 2, 5 and 7, and on day 8 drugs were removed by completely replacing the medium. Further cells were maintained by replenishing 30 % conditioned culture medium on days 12, 15, 19, 22. In the later days, when the cells began to reach confluence they were split 1:4. Cells were sampled over a 4-week period post-treatment until regrowth. For the cells treated with 50 µM vemurafenib, samples for RNA-seq were taken at days 2, 5, 8, 12, 19 and 28 after treatment; for 10 µM - at days 2, 8, 14 and 22. Experiments were performed in triplicate. RNA isolation was performed using AllPrep DNA/RNA/miRNA Universal Kit (50) (Qiagen, Germany). RNA quality was determined using RNA 6000 Pico Kit and Agilent 2100 bioanalyzer (Agilent Technologies, USA). Transcriptome library preparation was performed using the MGIEasy RNA Directional Library Prep Kit (MGI, China). Veriti 96 Well Thermal Cycler (Applied Biosystems) was used to carry out all reactions and incubations as intended in the library preparation protocol. RNA enrichment was performed by depleting rRNA with MGIEasy rRNA Depletion Kit (MGI, China). Work continued accordingly with instructions to the 250bp insert size. 