GSM4155928: Hair follicle mesenchyme from 2mo mice - replicate 2; Mus musculus; RNA-Seq
Backskin from α-SMACreT2:RosaYFP mice at 2 months of age and 18 months of age (n = 2/ sample) were surgically removed. Isolated backskin were incubated in 5mg/mL dispase (StemCell Technologies) in Hank's Buffered Salt Solution (HBSS; Life Technologies) for 30 mins at 37oC, then the epidermis was surgically removed. The remaining dermis was dissociated in 2mg/mL collagenase in Ham's F12 Nutrient mixture (Thermo Fischer Scientific) for 2 hours at 37oC. Cell suspension was diluted with cold HBSS, strained through 70µm and 40µm cell filters (Falcon) and centrifuged at 280g. Re-suspended cells were stained with conjugated Integrin α-9-PE (ThermoFisher), conjugated CD200-PE (ThermoFisher) and conjugated Cd26-647 (R&D Systems) for 45 mins, then with corresponding Alexa Fluor secondary antibodies (Invitrogen) for 30 mins. eFluor 780 viability dye (eBioscience) was included in the last 5 mins to exclude dead cells and debris. FACS-isolation was performed using a FACSAria III (BD Biosciences) and analyzed using FCS Express 6 software. Single colour and appropriate isotype controls were used for compensation and gating. Libraries were prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v2 and v3 Chemistry. Single cells were sorted into 0.1% BSA–HBSS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample. We recovered 2727 and 1906 cells for Samples 1 and 2, respectively, with an estimated doublet rate of ≈3%. Based on this, we determined lane distributions for sequencing using Illumina HiSeq 4000 PE (75 bp paired-end reads) with a targeted sequencing depth of 100,000 reads/cell.