title: RNA-seq of hiPSCs adapted to homemade weekend-free media
design: RNA-seq of hiPSCs adapted to homemade weekend-free media
library_construction_protocol: WTC11 cells at 70-80% confluency were washed with PBS without calcium and magnesium ions, then treated with 0.5 mM EDTA in PBS in 37°C for 2 min. EDTA was aspirated and cells were detached and suspended in DMEM/F12. The cells were then centrifuged at 100g for 5 min, supernatant was aspirated and the cell pellet was suspended in 350µl of Lysis Buffer from quick-RNA Mini Prep kit fro Zymo Research (R1055). WTC11 cells were thawed and cultured for 1 passage to acclimate. A day after the passage, cells that were supposed to be adopted to the medium were fed a 1:1 mixture of commercial E8 and weekend-free medium (homemade E8 or B8). The day after, the medium was replaced with a full volume of weekend free medium. From that point, cells were kept and passaged in a given weekend-free medium. In parallel, cells from the same vial were also kept in commercial E8. Cells were kept in culture and splitted for 5 passages and then harvested for mRNA extraction. total mRNA was extracted with quick-RNA miniprep kit from Zymo (R1055). Quality assessment and quantification was evaluated with Agilent Tapestation RNA kit (5067-5576 and 5067-5577). After quantification, the RNA was diluted to perform the library preparation on 500ng of RNA. Library construction was made by following the manufacture instruction of the Illumina Stranded mRNA Prep Ligation kit (20040534 and 20040553).