GSM7434218: Tree shrew2; Tupaia belangeri; RNA-Seq
The entire brains of the tree shrew embryos were immediately transferred to ice-cold DMEM solution with 10% FBS (DMEM/FBS) after dissection. Centrifugation of the samples was performed at 4°C. After transferring the whole brains of tree shrew embryos to ice-cold DMEM solution with 10% FBS (DMEM/FBS), the dura mater and blood vessels were removed from the brains. Brain tissues were dissociated using a modified protocol from the Worthington Papain Dissociation System. In brief, the dissected brains were cut into small pieces of less than 1mm3. The small pieces were then digested with 1mg/ml papain and 0.4% DNaseI at 37°C for 30min, shaking once every 5 minutes. After 40 minutes, the digestion process was blocked with DMEM/FBS. Cell suspensions were filtered through a 100 µm cell strainer and pelleted in a benchtop centrifuge at 200×g for 5 minutes. Pellets were re-suspended in DMEM and strained through a 40 µm filter.    To remove cell debris, 2ml cell suspension were layered onto a gradient composed of 1ml layers of 11%, 20% and 35% Optiprep1.32 pH 7.4 in DMEM and centrifuged for 5 min at 430×g with no brake. The bottom 1ml of the gradient was collected and mixed with 1ml DMEM. The cells were then pelleted at 550×g for 5min before being re-suspended in 100-200 µl DMEM with 0.2% BSA. The input cells were loaded onto the channel of Single Cell B Chip (v3 chemistry, PN-1000153). The 10x library was constructed using 10x Chromium Controller and Chromium Single Cell 3' Reagent Kits (v3 chemistry CG000183) following the detailed protocol of the manufacturer. In brief, single-cell suspensions in each channel of the chip were loaded on a Chromium Controller (10x Genomics, Pleasanton, CA) to generate single-cell GEMs (gel beads in the emulsion). The 10x libraries of each channel were then prepared using the Chromium Single Cell 3'Gel Bead and Library Kit v3 (PN-1000153, 1000075, 120262; 10x Genomics).