GSM7520156: Dural meninges IL-33KO, PND 14, 1; Mus musculus; RNA-Seq
P14 mice were anesthetized with CO2 and perfused transcardially with cold PBS. Horizontal cuts were made to remove the skullcap and placed in ice cold FACS Wash Buffer (FWB; 0.1M PBS (pH 7.4)/NaN3/3% FBS). Under a dissecting microscope, the dural meninges were quickly removed by gentle scraping using fine forceps and pulled away in an intact sheet. Meninges were kept in ice cold cRPMI ((RPMI 1640 supplemented with 10% (vol/vol) FBS, 1% (vol/vol) Hepes, 1% (vol/vol) Sodium Pyruvate, 1% (vol/vol) penicillin-streptomycin) until all samples were isolated. To obtain a single cell suspensions, meninges were transferred to cRPMI containing 40ug/mL DNAse I and 0.2 wU/mL Liberase TM in 1 mL of RPMI and incubated at 37 degrees Celsius with shaking at 220 RPM for 30 min. After gentle trituration, meninges and solution were passed through a 70 um cell strainer; remaining tissue was firmly pressed through the filter in a circular motion using a wide tip plastic plunger from a 1 ml syringe. The strainer was flushed with additional FWB and collected into a 15 ml falcon tube. Cells were pelleted at 300 RCF for 7 min at 4°C and resuspended in FWB for downstream applications. Cell suspensions were incubated with Fc block (2.4G2) and stained with the following antibody solution in FWB (45 min at 4 degree Celsius): CD45-BUV395, CD31-AF488, CD11b-PB, PDGFRa-APC, gp38-PECy7, DAPI. Cells were washed in FWB and fluorescence activated cell sorting was performed on a BD FACS Aria Fusion. Cells were gated for viable single cells and were enriched for lymphocyte and stromal populations by sorting CD45+CD11b- (non-myeloid immune cells) and gp38+CD31- (stromal cells) separately, and adding each of those populations to 25% of total cells (1:1:2 ratio of lymphocytes: stroma: total viable populations, Fig. S1A). Genotypes of mice sequenced were IL-33 deficient (Il33mcherry/mcherry) or heterozygous controls (Il33mcherry/+). Meninges from 5-8 mice were pooled per genotype and sex to obtain sufficient number of cells for sorting. Library was prepared according to the manufacter's instructions (Chromium Single Cell 3' v3 protocol, 10x Genomics).