title: GSM5640387: scRNA_seq_lambskin_straight; Ovis aries; RNA-Seq
library_construction_protocol: Hu sheep from Suzhou sheep breeding farm were used in this study. The experimental animals were healthy and under the same management. According to the wool length and curvature, wavy wool individuals and straight wool individuals were selected to obtain lambskin samples. After infiltration anesthesia through hypodermic injection of 2% lidocaine hydrochloride (10 mg), skin samples approximately 2 cm 2 and 2 mm deep were harvested from the body side of lamb. Subsequently, the wound was sewed up and the sample were divided into two parts, one was fixed with 4% paraformaldehyde (Solarbio, Cat#P1110) and another one was kept in DMEM/F12 medium (Gibco™, Cat#31331093) with penicillin-streptomycin (Gibco™, Cat#15240062) for subsequent analysis.To dissociate lambskin into single cells, skins from wavy wool individuals and straight wool individuals were pooled from each group. In order to ensure the quantity of hair follicle cells for each group, we adopt two strategies for dissociation. For strategies one, single hair follicles were isolated and dissociate into single cells. For strategies two, the whole skin was dissociated into single cells. Finally, single cells from two strategies were mixed as the final example. For hair follicles, skins were cut into small pieces and the hair follicles were isolated via microdissection using stereoscope. Collected hair follicles were incubated with pre-warmed (37 °C) TrypLE (Gibco™, Cat#12604013) for a total of 15 min in a 37 °C incubator on a shaker at 65 rpm for a gentle swirl. During the incubation, the dissociation mixture was agitated every 5 min with gentle pipetting. Then, cold 3% bovine serum albumin (BSA) solution (Sigma-Aldrich, Cat#A1933) was added to the dissociated cell suspension to inactivate TrypLE enzymatic activity. For skin tissues, 2 mg/ml collagenase IV (Sigma-Aldrich, Cat#C4-BIOC) was used to digest skin tissues at 37 °C for 30 min. Then, the products were incubated with pre-warmed (37 °C) TrypLE (Gibco™, Cat#12604013) for a total of 15 min. In this process, the skin tissues were mechanically dissociated into single cell suspension by pipetting. The cell suspensions were then filtered through a 40 μm nylon cell strainer (Sangon Biotech, Cat#12604013). Subsequent, the suspension from hair follicles and skins were mixed. An additional three washes with cold 3% BSA solution were performed. Cells were resuspended in 3% BSA. Viability (live cell percentage) and live cell count was determined using Trypan blue (used at a 1:1 ratio) and Countess II. The final cell concentration was around 1,000 cells/μl, with a cell viability greater than 90%. Single-cell RNA-seq libraries were prepared using Single Cell 3' Library Gel Bead Kit V3 (10x Genomics Inc., Pleasanton, CA, USA) following the manufacture's introduction.