title: GSM8798809: HS_P365_all_1; Mus musculus; RNA-Seq
library_name: GSM8798809
library_construction_protocol: 3-hour dissociation protocol Animals were anesthetized, shaved, depilated, and transcardially perfused with 50 mL PBS to remove any blood. Their skin was carefully dissected, and adipose tissue was removed with scalpels, supplemented by three successive washes in 30 mL RPMI. Each piece of skin was then carefully placed in a petri dish, epidermis side down, and treated with 15 mL of 0.15% trypsin-EDTA at 37°C for 30 minutes. After this incubation, the skins were removed from the trypsin-EDTA solution and placed in a PBS/5%FBS solution at 4°C, where the detached epidermis was gently scraped off with scalpels. The skins were then washed three times in 30 mL of RPMI before being exposed to the trypsin-EDTA medium at 37°C for another 30 minutes. After this second incubation, the same procedure was used to scrape off the enzymatically dissociated epidermis. After a final rinse with RPMI, the skins were carefully dissected under a Leica epi-fluorescence microscope to isolate cNFs and HLS regions, which were then finely minced on a -20°C metal block using scissors and scalpels. The minced tissue (0.5 g per tube) was then immersed in 10 mL of prewarmed RPMI medium containing Liberase DH (0.25 mg/mL) and placed in a 37°C water bath for 3 hours. To achieve complete dissociation, the skin tissue was mechanically dissociated every 30 minutes with a 1 ml pipette. After 40 minutes of dissociation, DNAseI was added to the medium. After the 3-hour incubation period, the dissociation tubes were immediately placed on ice. Any remaining skin pieces were then mechanically dissociated using an up-and-down motion, first with a 1 ml pipette and then with a glass Pasteur pipette pre-coated with FBS. To stop the action of the enzymes, 30 ml of RPMI containing 10% FBS was added to each tube. The tubes were then centrifuged at 300g for 10 minutes. After discarding the supernatant, the pellet was gently resuspended in 5 ml of RPMI and filtered through a 100 μm filter, followed by a 70 μm filter, and finally a 30 μm filter to remove any remaining tissue fragments or cellular debris. The cells were then centrifuged again at 300g for 10 min before being resuspended in a solution of PBS/1% BSA. To assess cell viability, we added DAPI viability dye at a concentration of 5 μg/ml. The prepared cells were then sorted using Fluorescence-Activated Cell Sorting (FACS) at the IMRB FACS facility. The sorting process was based on the cells' DAPI labeling and Tomato fluorescence to ensure the selection of viable and relevant cell populations. 18-hour dissociation protocol Animals were anesthetized, shaved, depilated, and transcardially perfused with 50 mL PBS to remove any blood. Their skin was carefully dissected, and adipose tissue was removed with scalpels, supplemented by three successive washes in 30 mL RPMI. Each piece of skin was then carefully placed in a petri dish, epidermis side down, and treated with 15 mL of 0.15% trypsin-EDTA at 37°C for 30 minutes. After this incubation, the skins were removed from the trypsin-EDTA solution and placed in a PBS/5%FBS solution at 4°C, where the detached epidermis was gently scraped off with scalpels. The skins were then washed three times in 30 mL of RPMI before being exposed to the trypsin-EDTA medium at 37°C for another 30 minutes. After this second incubation, the same procedure was used to scrape off the enzymatically dissociated epidermis. After a final rinse with RPMI, the skins were carefully dissected under a Leica epi-fluorescence microscope to isolate cNFs and HLS regions. Small pieces (2x2 mm) of each sample type were cut and then immersed (6 pieces per well) in 2 ml of DMEM medium containing 10% FBS, 10% penicillin/streptomycin, and Liberase DH (0.25 mg/mL). The filled 6-well plate was then placed into an incubator (37°C, 5% CO2) overnight for 18 hours without agitation. The following day, to stop the action of the enzymes, 8 ml of DMEM medium containing 10% FBS were added to each well, followed by gentle mechanical dissociation with a 1 ml pipette. The tubes were then centrifuged at 300g for 10 minutes. After discarding the supernatant, the pellet was gently resuspended in 5 ml of DMEM and filtered through a 100 μm filter, followed by a 70 μm filter, and finally a 30 μm filter to remove any remaining tissue fragments or cellular debris. The cells were then centrifuged again at 300g for 10 min before being resuspended in a solution of PBS/1% BSA. To assess cell viability, we added DAPI viability dye at a concentration of 5 μg/ml. The prepared cells were then sorted using Fluorescence-Activated Cell Sorting (FACS) at the IMRB FACS facility. The sorting process was based on the cells' DAPI labeling to ensure the selection of viable cell populations. All or Tom+ skin cells were sorted (BD InfluxTM Cell Sorter) using a DAPI nuclear labeling viability dye and the Tomato endogenous fluorescence. Cells were collected in 0.04% BSA-PBS solution. Around 25,000 cells were loaded into one channel of the Chromium system using the V3.1 single-cell reagent kit (10X Genomics) to generate single-cell GEMs. Following capture and lysis, cDNAs were synthesized, and then amplified by PCR for 12 cycles as per the manufacturer's protocol (10X Genomics). The amplified cDNAs were used to generate Illumina sequencing libraries that were each sequenced on one flow cell Nextseq500 Illumina.
identifiers.namespace: GEO
identifiers.id: GSM8798809_r1