title: GSM3767569: Injured; Mus musculus; RNA-Seq
library_construction_protocol: Proximal incisors (roughly 3 mm in length) were first severed away using a pair of scissors after removal of the surrounding jaw bones. The bulbous portion, as well as the lateral wing-shaped epithelium, and the surrounding mesenchyme were subsequently dissected from the rest of the incisor and collected in cold HBSS (pH 8.0) and digested enzymatically at 37°C in collagenase type II at concentration 2 mg/ml (Gibco/BRL type II in DMEM) for 30 min, followed by collagenase inactivation with 2% FBS. The digested tissue was mechanically disrupted using 1 ml pipette tip and centrifuged at 400 x g for 3 min. Cells were resuspended in HBSS (pH 8.0) containing 2% FBS. To isolate epithelial cells by fluorescence-activated cell sorting, proximal incisor cell suspension was stained with mAb specifically recognizing CD326 (G8.8, AF647 conjugate, Biolegend) for 30 min on ice followed by centrifugation at 400 x g for 3 min. Prior to fluorescence-activated cell sorting (BD FACSAria2 SORP), cells were filtered through 40 μm mesh (BD Falcon) and stained with DAPI. Epithelial cells from 5 controls and 5 5FU-treated mice were isolated as described above. The resulting cell suspensions (~20,000 cells each) were submitted as separate samples with targeted cell recovery of 3,000 per sample to be barcoded for single-cell RNAseq using the Chromium Controller (10X Genomics) and the Single Cell 3' Library Kit v2 (PN-120236/37/62). Resulting libraries were sequenced on a HiSeq 4000 (Illumina) using HiSeq 4000 PE Cluster Kit (PN PE-410-1001) with HiSeq 4000 SBS Kit (150 cycles, PN FC-410-1002), one sample being loaded per sequencing lane.