GSM6397216: day2_ctrl_050; Gallus gallus; RNA-Seq
BP specimens were incubated in prewarmed 500 µg/ml thermolysin in medium 199 at 37℃ for 30 min. To stop enzymatic dissociation, specimens were transferred into medium 199 supplemented with 5% FBS. Afterwards, the BP was separated from the surrounding tissues under the stereomicroscope. Then, the samples were transferred into an Eppendorf tube by a pipette. After centrifugation (800g x 5 min) at room temperature, the supernatant was removed. Samples were incubated in an enzyme cocktail containing 50% Accutase and 50% TrypLE Select enzyme for 7 min at 37℃. After gently pipetting 10-20 times, samples were incubated for additional 4 min at 37℃. After inactivation of enzymatic activity with medium 199 supplemented with 5% FBS, further mechanical dissociation by pipetting was performed. Dissociated cells were collected after centrifugation and resuspended in medium 199 supplemented with 5% FBS. Capture of the single cells and cDNA synthesis were conducted using C1 Single-Cell mRNA seq HT IFC for medium cell size (10-17 µm) and Reagent kit v2 (Fluidigm), according to manufacturer's instruction. Briefly, 12,000 cells of each sample were loaded onto HT IFC, and 400 cells were captured in the IFC. Sequencing libraries were prepared by transposase-assisted tagmentation and enrichment of 3'end of genes using Nextera XT DNA Library Prep Kit (Illumina). The libraries were sequenced on the HiSeq2500 for 25 and 75 cycles paired-end reads.