title: 1772117132-C08
organism: Mus musculus
description: Protocols: Mouse work: Lgr5-EGFP-Ires-creERT2, Lgr6-EGFP-Ires-creERT2 and R26R-tdTomato knock-in strains were described previously. All experiments were performed on young adult females of C57BL/6 background. Unless stated differently, tracing of Lgr5-EGFP and Lgr6-EGFP cells was induced by intraperitoneal injection of 2 x 3 mg of Tamoxifen (1 day apart) at around 7 weeks of age and at least one week prior to wounding. Two 4 mm diameter full thickness wounds were created on the dorsal skin of anesthetized mice at 8 weeks of age by holding up a skin fold and piercing it on both sides with a dermatological biopsy punch. Wounds were allowed to heal for 1 day, 4-5 days, 7-8 days, 10-12 days and more than one month before mice were carefully sacrificed by cervical dislocation without tearing the wounds. In addition, unwounded controls were taken at the same age (0-day control). All animal experiments were performed in accordance to Swedish legislation and approved by the Stockholm South Animal Ethics Committee. Cell isolation: Full epidermal cells were isolated as described previously (Jaks et al., 2008) with minor changes to the protocol. Before cell isolation commenced, brightfield and fluorescence pictures of all wound were taken using a Zeiss Discovery v20 stereomicroscope. The complete dorsal skin including the wound sites was then carefully removed, floated on ice-cold HBSS and small circles of skin were cut from around the two wound sites (wound area) and from unwounded skin (control area) using a 12-mm dermatological biopsy punch. Next, dermal and adipose tissue was carefully scraped off using a scalpel with special attention to not losing wound front, wound epidermis or wound matrix cells. The stripes of skin were subsequently floated on trypsin (Sigma) for 2h at 32°C, epidermal tissue and wound matrix was scraped into S-MEM / 1% BSA and a single cell suspension was obtained by carefully pipetting up and down. The resulting cell suspension was filtered through 70 µm and 40 µm cell strainers, resuspended in Defined Keratinocyte Serum-free Medium without supplement (DK-SFM) (Life Technologies) and transferred to 5 ml FACS tubes coated with poly-HEMA. Tomato-traced cells were sorted using a FACSAria III (BD). Thresholds were set by analysing R26R-tdTomato-negative Lgr5-EGFP-Ires-creERT2 or Lgr6-EGFP-Ires-creERT2 mice and then maintained over all experiments. For each experiment, wound area and control area cells were captured on 3:1 medium microfluidic chips (designed for cells from 10 µm - 17 µm) using several Fluidigm C1 Autoprep systems in parallel. Ideally, a concentration of >500 cells / µl in DK-SFM with DNase I was used for capturing. Capturing efficiency and cell quality was evaluated on a Nikon Eclipse Ti-U automated microscope and both brightfield and Cy3 images of every capturing position were taken. Only single, healthy looking, Tomato-positive cells proceeded to library preparation. Single-cell cDNA synthesis, tagmentation and isolation of 5' fragments, Illumina high-throughput sequencing and processing of sequencing reads was performed as described previously (Joost et al 2016). All single-cell experiments were performed with the help of the Eukaryotic Single Cell Genomics core facility at SciLifeLab, Stockholm. Library preparation from single-cell cDNA was performed using a tagmentation-based approach as described in detail in Islam et al., 2014.