single-cell RNA-seq of mouse back skin treated with TPA
1-5 mm3 biopsies from shaved mouse back skin were incubated with either: I) dissociation buffer (1.25 mg/ml collagenase I (Sigma), 0.5 mg/ml collagenase II (Worthington), 0.5 mg/ml collagenase IV (Worthington) and 0.1 mg/ml hyaluronic acid (Sigma) in Hanks Balanced Salt solution (HBSS) (Invitrogen)) for 2.5 hours followed by addition of trypsin (Santa Cruz Biotechnology) to a final concentration of 0.25% for 30 minutes, or II) 0.25% trypsin (Santa Cruz Biotechnology) for 2 hours followed by incubation with dissociation buffer for 1 hour. All dissociation steps were performed at 37°C and shaking at 600 rpm. After 3 hours enzymatic digestion was quenched with HBSS supplemented with 10% fetal bovine serum (FBS, Life Technologies) and cell suspensions filtered through 40 µm cell strainers (Grenier Bio-One). Strainers were washed with an additional 2-3 ml HBSS containing 10% FCS and cells pelleted by centrifugation at 300 x g for 8 minutes at 4°C. Cell pellets were carefully (using wide-bored P1000 pipet tips) resuspended in 100-200 μl sorting buffer (1 mM EDTA (Invitrogen), 0.04% HyClone bovine serum albumin (BSA, GE Healthcare Life Sciences) in PBS). Cell suspensions were either passed through a 40 µm FLOWMI cell strainer (Fisher Scientific) and viability and concentration determined using 0.4% trypan blue stain (Invitrogen) and Countess II automated cell counter (Invitrogen) or subjected to Fluorescence-activated Cell Sorting (FACS).    Cell pellets for FACS were resuspended in sorting buffer containing 1 μg/ml propidium iodide (PI) (Life Technologies) and transferred to low-binding polypropylene FACS tubes. Live cells from each sample were sorted into one 96-well flat-bottom low attachment tissue culture plate well containing 30 μl sorting buffer on a cold plate using Aria II flowcytometry cell sorter (BD Biosciences) with a 70 µm nozzle (70 psi). PI was excited with a 561 nm laser and fluorescence was recorded with a 610/20 595LP filter. Cells were gated using; i) FSC-A vs SSC-A to remove debris ii) SSC-W vs SSC-A to exclude doublets iii) RL780/60-A vs PI-A to exclude auto-fluorescent cells and dead cells (PI positive). Samples were handled on ice whenever possible. 10X Genomics Chromium Single Cell 3' cDNA libraries were prepared according to the manufactures' instructions. Single cells were encapsulated into droplets in the Chromium Controller instrument for cell lysis. Single cells were encapsulated into droplets in the Chromium Controller instrument for barcoded reverse transcription (RT) of mRNA, followed by amplification, shearing and Illumina library construction. Quantification of the libraries was carried out using the Qubit dsDNA HS Assay Kit (Life Technologies), and cDNA integrity was assessed using D1000 ScreenTapes (Agilent Technologies).