title: GSM8681926: Dura mater, sham surgery; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized and perfused with 20 mL of PBS. Skullcaps were dissected using small surgical scissors and immediately placed in ice-cold PBS. Dural meninges were carefully peeled off the skullcaps using fine forceps and transferred to tubes for enzymatic digestion in RPMI medium (Gibco, 21875-034) supplemented with 2.5 mg/mL collagenase D (Roche, 11088866001) and 0.1 mg/mL DNase I (Roche, 11284932001) for 20 minutes at 37°C. The tissue was then dissociated by repeated pipetting (25 strokes) and passed through a 35-μm cell strainer. Following centrifugation (5 minutes at 400g, 4°C), cells were resuspended in 150 μL PBS and pooled from two meninges per well in 96-well V-bottom plates. To minimize nonspecific antibody binding, cell preparations were first incubated for 10 minutes at 4°C with anti-CD16/32 (Fc block; BD Biosciences, 5 µg/mL). Subsequently, a fluorescently conjugated antibody cocktail, including Live-or-Dye 405/452 (Biotium, 32003, 0.05 µL/test), was added. Cells were incubated with the antibodies for 30 minutes at 4°C, followed by centrifugation (5 minutes at 500g, 4°C) and a single PBS wash. Finally, cells were resuspended in 600 µL of PBS and sorted using the MoFlo Astrios cell sorter (Beckman Coulter). Dural cells were identified as viable singlets and collected in PBS containing 20% BSA. singleCell library prep were realized with chromium from 10X Genomics. Chromium Next GEM Single Cell 3' GEM kit and Chip G were used, following manufacturer's recommendations. 20000 cells were loaded on the chromium and sequenced on ILLUMINA NovaseqXplus.