GSM9401503: FLEX-Pool1-P12; Mus musculus; RNA-Seq
Brains were placed in a chilled brain slicer (WPI; rodent brain matrices acrylic) to obtain 1mm thickness of coronal brain sections. According to Allen brain atlas reference, isocortex and thalamic area were microdissected using microscalpel (WPI, 500250) under microscope in ice cold dissociation medium solution (DM; Hank's balanced salt solution without calcium and magnesium (Thermofisher, 14175095), 10mM HEPES (Thermofisher, 15630080), 9mM MgCl2 (Fisher Scientific, AAJ61014EQE), 35mM D-glucose (Fisher Scientific, AAJ60067AK), pH=7,35). After slicing in little pieces, brains were enzymatically digested using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130-092-628) for 15 min in mix 1 and 10 min in mix 2 at 33°C. During the digestion process, tissues were homogenized two times by up and down pipetting. At the end of the digestion process, samples were put in the ice with DM solution and cell clusters were removed by filtration through prewetted 70um and centrifuge at 200g for 8 min at 4°C. To stop the enzymatic reactions, the pellet is resuspended in DM solution with 3mg of ovomucoid protease (Worthington, LK003182, resuspended in DM solution) and 300 units of DNAse1 (Worthington, LK003172, resuspended in DM solution). The myelin and the cell debris were removed by using the debris removal solution (Miltenyi Biotec, 130-109-398). After 10 min blocking at RT with 5ug/mL of CD16/CD32 Fc Block (eBiosciences, 16-0161-82), the cells were stained at RT for 20 min with APC anti mouse CD31 (BD Biosciences, 551262) and live/dead fixable Aqua dead (ThermoFisher Scientific, L34957) allowing the staining of dead cells. After staining process, the cell pellet is fixed between 16h and 20 hours at 4°C according to the manufacturer's instructions (10x Genomics, Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit). After a washing step, the stained cells were resuspended with 2mM of EDTA and 0,04 % of BSA. The setting up of the gates and compensations were allowed with the unstained cells, the single positive staining, the Fluorescence Minus One (FMO) and the isotype control staining (APC Rat IgG2a, κ (BD Biosciences, 551139). After removing the dead cells and doublets, endothelial cells (CD31+) were sorted using a BD FACS Aria Fusion from 26 P6, 19 P12 and 39 2-month-old mice and stored at -80C in buffer. Single-cell suspensions of fixed and sorted brain cells from P6, P12 and adult Thalamus and Cortex regions (12 datasets in total) were resuspended in PBS containing 0.04% ultra-pure BSA. Multiplexed libraries were prepared using the Chromium Next GEM Single Cell Fixed RNA Kit, Mouse Transcriptome, 4rxns x 4 BC (# 1000496, 10x Genomics; Pleasanton, CA, USA) according to the manufacturer's instructions. The target recovery for each sample was 10,000 cells.