GSM5703291: Organoid_day_30_Dex_Veh_rep1; Homo sapiens; RNA-Seq
Single cells were dissociated using StemPro Accutase Cell Dissociation Reagent (Life Technologies), filtered through 30uM and 20uM filters (Miltenyi Biotec) and cleaned of debris using a Percoll (Sigma, P1644) gradient. Single cells were resuspended in ice-cold Phosphate-Buffered Saline (PBS) supplemented with 0.04% Bovine Serum Albumin and prepared for single-cell separation. All experiments were performed in quadruplicate at all three time points, with a paired design of a treatment (Dex) or control (Veh) condition per each experiment/iCell8 chip, for a total of 12 chips, using the iCELL8 Single-Cell System (Wafergen, Takara Bio) according to the manufacturer's recommendations. Briefly, cells were stained with Propidium Iodide (for dead cells) and DAPI (for live cells) on ice for 5-10 minutes and dispensed in the loading plate. Following microfluidic separation, iCell8 chips were imaged using the built-in fluorescence microscope and snap-frozen and stored at -80C until library preparation. Single-cell sequencing was performed according to the manufacturer's guidelines using the iCell8 Chip and Reagent Kit using in-chip RT-PCR amplification chemistry (Wafergen, Takara Bio). On each chip, 2 samples were loaded in parallel (treatment and vehicle condition) and subsequenctly, based on fluorescence labeling, only wells containing live single cells were selected for library preparation. All libraries were prepared using the Nextera XT DNA Library Preparation Kit and Nextera XT Index Kit (Illumina). Sequencing was performed on an Illumina HiSeq4000 (Illumina, San Diego, CA), 1 lane per chip, sequencing paired-end with 26nt/100nt configuration.