title: GSM6138447: E9.5 mand, scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: E12.0 mandibular arches from K14Cre;R26mT/mG embryos were dissected in cold HBSS. Tissues were then pooled and incubated with 10 mg/ml Dispase II in HBSS supplemented with 10 µg/ml DNase at 37°C and swirled at 100 rpm for 32 minutes to enzymatically separate the epithelium from the mesenchyme. Epithelia were peeled off using forceps and dissociated in TrypLE at 37°C for 30 minutes, with gentle pipetting every 10 minutes. Cells were then centrifuged at 400 rcf for 5 minutes and resuspended in cold flow cytometry buffer (calcium free HBSS with 5% fetal bovine serum (FBS), 2 mM EDTA, and 10 mM HEPES). Undissociated cell clumps were sieved out using a 20 µm pluriStrainer. The resulting cell suspension was sorted using fluorescence-activated cell sorting (FACS) to isolate GFP+ single epithelial cells. To collect E9.5 mandibular cells, mandibular arches were dissected in cold HBSS, washed with DPBS without Ca2+/Mg2+, and directly incubated in TrypLE at 37°C for 19 minutes with shaking at 300 rpm. Tissues are gently pipetted up and down for 4 times at the 12th minute mark. Cells were then spun down at 250 rcf for 4 minutes at 4°C and resuspended in DPBS with 0.5% BSA. Undissociated cell clumps were sieved out using a 20 µm pluriStrainer. Cell numbers and viability of dissociated cells were analyzed using the Invitrogen Countess II FL. Library was performed using 10x Genomics single cell 3' V3 chemistry according to the manufacturer's instructions. Briefly, dissociated cells were combined with barcoded Gel Beads and Partitioning Oil onto a Chromium Chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retro-transcribed to cDNA using primers containing the TruSeq Read 1 (R1) primer sequence, the 10x Barcode, and a Unique Molecular Identifier (UMI). The pooled barcoded cDNA was then cleaned up using Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected using SPRIselect for subsequent library construction. During the library construction, the TruSeq Read 2 (R2) primer sequence, the P5 and P7 paired-end construct primer sequences and a sample index were added via End Repair, A-tailing, Adaptor Ligation, and PCR.