GSM6449999: WT, LPS [MGISEQ]; Mus musculus; RNA-Seq
For single cell dissociation, artificial cerebral spinal fluid (ACSF) was prepared using the “protective cutting” formula. Before the experiment, ACSF was bubbled with carbogen gas (95% O2 and 5% CO2) for ~30 mins. Mice were anesthetized and perfused transcardially with ACSF. The brain was quickly removed and submerged in carbogen bubbled ice-cold ACSF. 8 brain slices (200μm) were sectioned using vibratome and the motor cortex was micro-dissected. The dissected tissue pieces were then digested using the Papain dissociation system, supplemented with DNaseI. After 30 mins, the digestion was quenched by exchanging the solution with quenching (Q) buffer (ACSF with 1% FBS and 0.2% BSA) 3 times. The quenched solution was placed on ice for 5 mins and then proceeded to trituration. The tissue pieces were gently triturated through Pasteur pipettes with fire polished tip openings at 3 different diameters (600μm, 300μm, 150μm). The cell suspension was then centrifuged at 100g for 10 mins and resuspended in Q buffer for FACS sorting. Single cells were sorted (BD Aria III) using a 130μm nozzle and a sheath pressure of 10 psi. 15mL conical tube containing ~12mL collection (C) buffer (ACSF with 13.2mM trehalose, 2% FBS and 1% BSA) was used for collecting the target population. Library construction was performed according to the manufacturer's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, dissociated brain cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA. For each library, cDNA were amplified with polymerase chain reaction (PCR). The purified product were then converted to DNA nanoballs (MGIEasy Universal Library Conversion Kit) for sequencing with the MGISEQ-2000 or DNBSEQ-T7 model.