title: GSM1650724: 1772067078_H11; Mus musculus; RNA-Seq
library_construction_protocol: Clipped and disinfected dorsal skin was isolated, dermal and adipose tissue was removed and strips of skin were floated on trypsin (Sigma) for 2h at 32°C.  Epidermal tissue was subsequently scraped into S-MEM / 1% BSA and single cells were isolated by magnet stirring at 120 rpm for 25 min / RT. The resulting cell suspension was filtered through 70 µm and 40 µm cell strainers, resuspended in in Defined Keratinocyte Serum-free Medium (DK-SFM) without supplement (Life Technologies) and Sca1+ and Sca1- were separated using Anti-Sca1-FITC magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were stored on ice in DK-SFM with 0.1 mg/ml DNase I (Stem Cell Technologies) until capturing. Before capturing, the cell suspension was carefully resuspended and two times passed through a 20 µm cell strainer. Epidermal cells were captured on a medium microfluidic chip (designed for cells from 10 µm – 17 µm) using the Fluidigm C1 Autoprep System. 14 µl filtered cell suspension (about 750 cells / µl in DK-SFM with DNase I) was mixed with 6 µl C1 Suspension Reagent and 14 µl were loaded onto the chip. Single-cells were then captured for 30 min at 4°C using the “Cell Load (1772x/1773x)” script. Lysis mix, RT mix and PCR mix (described in Islam et al., Nat Methods. 2014 Feb;11(2):163-6) were added to the chip. The plate was placed in the Fluidigm C1 instrument and the 'mRNA Seq: RT + Amp (1772x/1773x)' script was executed, and included lysis, reverse transcription and 21 cycles of PCR. The amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent. Amplified cDNA was simultaneously fragmented and barcoded by tagmentation using Tn5 DNA transposase to transfer adaptors to the target DNA as described (Ibid.). 100 µl Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were washed in 2× BWT (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and resuspended in 2 ml 2× BWT. Twenty microliters of beads were added to each well and incubated at room temperature for 15 min. All fractions were pooled, the beads were immobilized and the supernatant removed. The beads were resuspended in 100 μL TNT (20 mM Tris, pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen Qiaquick PB, and twice in 100 μL TNT. The beads were resuspended in 100 μL restriction mix (1× NEB CutSmart, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3′ fragments carrying a PvuI recognition site. The mix was incubated for 1 h at 37 °C, then washed three times in TNT. Finally, to elute DNA, beads were resuspend in 30 µL ddH2O and incubated 10 minutes at 70°C. Beads were then immediately bound to magnet and the supernatant was collected. To remove short fragments,  Ampure beads (Beckman Coulter) were used at 1.8× volume and eluted in 30 µL.