GSM6543166: mutant mouse brain 1, supratentorial, Smarcb11148del/1148del; Mus musculus; RNA-Seq
Isolated brains from two Smarcb11148del/1148del and two Smarcb1+/+ P0 mice were divided into a supratentorial and an infratentorial part and each minced with scalpels on ice. For enzymatic dissociation, 2-3 mL of a solution composed of 1 vial Papain (Worthington), 5 mL pre-equilibrated DMEM:F12 and 34 µL DNAse I diluted in 250 µL DMEM:F12 were added to each sample, followed by incubation at 37 °C in 5 % CO2 for 30 min. Afterwards, the cell solution was transferred onto a 40 µm cell strainer. The reaction was stopped with 2 mL PBS / 10 % BSA before washing with 10 mL PBS. The obtained single-cell suspensions were stained with 7-Aminoactinmycin D (7-AAD) (eBioscience). Non-viable, 7-AAD-positive cells were removed by Fluorescence-activated cell sorting (BD FACS Aria II), followed by manual counting with Trypan Blue staining. Approximately 25,000 vital single-cells of each sample were processed for single-cell RNA sequencing using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1, the Chromium Next GEM Chip G and the Single Index Kit T Set A (10X Genomics) according to the manufacturer's instructions. In brief, single-cell GEM (Gel Beads-in-Emulsion) were generated by the Chromium Controller, followed by GEM-RT (reverse transcription), Dynabeads cleanup, cDNA amplification, SPRIselect beads cleanup and Library construction. Quality, purity, size and concentrations of cDNA and libraries were assured by measurement on a Tapestation 2000 (Agilent Technologies). Indexed libraries were sequenced separately by the Illumina NextSeq 500 sequencing platform (v 2.5 chemistry, 75 cycle kit). For all samples, the same library was sequenced a second time to increase sequencing saturation.