title: GSM8395003: CT 8-weeks old, control mice, pool of 3 mice; Mus musculus; RNA-Seq
library_name: GSM8395003
library_construction_protocol: Cell suspensions from adult mouse skin were obtained through mechanical dissociation and enzymatic digestion. Briefly, adult back skin from W8 or W24 mice was harvested in pre-digestion medium, finely minced and incubated at 37°C on a rotating plate to remove epithelial cells and other impurities. The samples were then digested for 45 minutes on a rotating plate with 1.25 mg of Liberase TM (Sigma) and 2.5 mg of DNAse I (Sigma) to disaggregate the tissue. Samples were further dissociated with the Miltenyi gentleMACS Dissociator and cells were then enriched with a Percoll gradient. Cell suspensions from mouse skin were blocked with anti-mouse CD16/32 (S17011E, Biolegend) for 15 min at 4°C. Surface staining using CD45 was performed in FACS buffer for 1 hour at 4°C and CD45+ immune cells were sorted using FACS. Approximately 16,000 cells per sample were encapsulated into droplets using Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 according to manufacturer's protocol (10× Genomics), Briefly, after generation of nanoliter-scale Gel bead-in-EMulsions (GEMs) using Next GEM Chip G, GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (BioRad) programed at 53°C for 45 min, 85°C for 5 min, and held at 4°C. After reverse transcription, single-cell droplets were broken and cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermofisher Scientific). cDNA was then amplified with a C1000 Touch Thermal Cycler programed at 98°C for 3 min, 12 cycles of (98°C for 15 s, 63°C for 20 s, 72°C for 1 min), 72°C for 1 min, and held at 4°C. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, index adaptor ligated, and cleaned with cleanup mix containing SPRIselect Reagent Kit (Beckman Coulter) in between steps. Post-ligation product was amplified and indexed with a C1000 Touch Thermal Cycler programed at 98°C for 45 s, 14 cycles of (98°C for 20 s, 54°C for 30 s, 72°C for 20 s), 72°C for 1 min, and held at 4°C. The sequencing - ready libraries were cleaned up with SPRIselect beads. 10x libraries were pooled and charged with 1% PhiX on one SP lane of the NovaSeq 6000 instrument (Illumina) using the NovaSeq 6000 SP Reagent Kit v1.5 (200 cycles), and the following sequencing parameters: 28 bp read 1 – 8 bp index 1 (i7) – 150 bp read 2. The SP lane generated a total of 434.10^6 reads.