Pluto Bioinformatics

GSE79374: Single-cell RNAseq of mouse brain tissues across 3 developmental time points

Bulk RNA sequencing

Brains were harvested from 2-month-old adult, p12 day old mice and e18.5 day mice embryo. Brain regions of interest (Visual cortex, Thalamus and Hippocampus) were dissected from fresh brains under a light microscope.; The tissues were enzymatically dissociated by incubating in 5 ml of a papain solution containing Earles balanced salts (EBSS, Sigma, St. Louis, E7510), D(+)-glucose (22.5mM), NaHCO3 (26mM), DNase (125U/ml, Worthington, Lakewood, LS002007), papain (9 U/ml, Worthington, Lakewood, NJ, LS03126), and L-cysteine (1mM, Sigma, St. Louis, C7880) at 35 C (incubation time varied with developmental stage: 60 minutes for adult and post-natal tissues, 30min for e18.5). The papain solution was equilibrated with 5% CO2 and 95% O2 gas before and during papain treatment. Following papain treatment, the tissue was mildly dissociated using the gentleMACS dissociator (Miltenyi Biotec, Germany), followed by gentle trituration using a 5ml pipette. The enzymatic reaction was stopped by adding 5ml of inhibitor buffer (Ovomucoid Trypsin inhibitor, Worthington, Lakewood, LK003182) and centrifuged at 130g for 5 minutes. The cell pellet was then resuspended in 10 ml RPMI1640 medium (Sigma, St.Louis, 11875093) containing 0.02% BSA and 12.5U/ml DNase and strained through a 37um filter (STEMCELL Technologies, BC, 27215) to remove undissociated cell clumps. This cell suspension was then depleted for debris using the Dead cell removal kit (Miltenyi Biotec, Germany, 130-090-101) according to manufacturers instruction. Live single cells were enriched by FACS sorting cells (LIVE/DEAD cell viability assay; Molecular Probes, Life Technologies). Single cells were counted and diluted to 250-500 cells per microliter.; Cells were loaded, captured on onto C1 integrated fluidic circuits IFC (5- to 10-m chip) for cell lysis, reverse transcription with oligo (dT) primers and amplification of cDNA on a C1 Single-cell Auto Prep System according to the mRNA-seq protocol of the manufacturer (Fluidigm, USA). After cells were captured on the chip, they were imaged using phase-contrast and fluorescence microscopy before cell lysis. Resulting cDNA was quantified with a Quant-iT PicoGreen dsDNA Assay Kit (PN P11496; Life Technologies), and quality was checked with High Sensitivity DNA Reagents (PN 5067-4626) according to the manufacturer's instructions (Agilent Technologies). Only cells with high-quality cDNA, with a concentration higher than 0.05ng/ul were processed for subsequent library preparation. Library preparation was performed using Nextera XT DNA Sample Preparation Kit (Illumina) as described in the Fluidigm manual. Following library preparation, cells were pooled and sequenced on an Illumina HiSeq 2500 platform as 1x100bp paired-end reads. SOURCE: Debarka Sengupta (debarka@gmail.com) - Genome Institute of Singapore