GSM4844520: E13epi; Mus musculus; RNA-Seq
Mouse submandibular salivary glands were isolated from 13-day mouse embryos as previously described (Sequeira et al., 2013). Briefly, a scalpel (Fine Science Tools, 10011-00 and 10003-12) was used to decapitate the mouse embryo. While the detached head was held on its side with one prong of forceps (Fine Science Tools, 11251-20) pierced through the top, a scalpel was used to slice across the mouth opening to isolate the mandible and tongue, between which the submandibular glands were sandwiched. Under a dissecting microscope, the detached mandible tissue was placed on a glass plate with the tongue facing down. A pair of forceps was used to slice through the midline of the mandible tissue to expose the tongue and the two submandibular glands attached to the base of the tongue. After surrounding tissues were removed, glands were detached using forceps and collected into a 35-mm dish with 3 mL DMEM/F-12 (Thermo Fisher, 11039047) media until all embryos were dissected. To isolate the epithelial rudiments, 4 to 6 glands (see Salivary gland isolation and culture) were treated with 150 µL 2 units/mL dispase (Thermo Fisher, 17105041; diluted in DMEM/F-12) in a well of a Pyrex spot plate (Fisher Scientific 13-748B) for 15 min at 37°C. The glands were washed twice with 5% BSA (MilliporeSigma, A8577; diluted in DMEM/F-12) in the same well to quench the dispase activity. The mesenchyme of each gland was removed using a pair of forceps (Fine Science Tools, 11254-20) and a tungsten needle (Fine Science Tools, 10130-05 and 26016-12) under a dissecting microscope. The forceps were mostly used to hold the gland still, whereas the needle was gently inserted between the mesenchyme and epithelium to separate them. Isolated epithelial rudiments were transferred to a new well of the spot plate with 150 µL DMEM/F-12 media using low-retention pipette tips (cut for larger opening; Rainin, 30389190) for temporary storage until all glands were processed. A total of 8 isolated epithelial rudiments were collected and rinsed in 1 mL HBSS (Thermo Fisher, 14170161) in a 2 mL protein LoBind tube (Eppendorf, 022431102). After the rudiments were pelleted by centrifugation at 100× g for 30 seconds, liquid was removed as much as possible using a 1 mL pipette followed by a 200 µL pipette under a dissecting microscope to avoid accidentally discarding the samples. 100 µL Accutase (Innovative Cell Technologies, AT104) was added to the tube, which was immediately incubated for 2 minutes in a 37°C water bath to disrupt cell-cell adhesion. While being monitored under a dissecting microscope, the epithelial rudiments were triturated using a 200 µL pipet set at 50 µL using a low-retention tip (Rainin, 30389187) for 2 min, when most cells were clearly dissociated. 900 µL 10% fetal bovine serum (FBS; GE Healthcare/Cytiva, SH30070.03) diluted in PBS was added to quench the Accutase. The 1 mL cell suspension was passed through a 40 µm Flowmi (VWR, H13680-0040) cell strainer into a polyethylene terephthalate (PET) 15 mL tube (Corning, 430053). It is critical to use PET tubes instead of polypropylene tubes for efficient cell recovery. Cells were pelleted by centrifugation at 100× g for 2 minutes in a swinging-bucket rotor. After the supernatant was carefully removed, cells were resuspended in 1% FBS in PBS and pelleted again. About 50 µL liquid was retained to resuspend the cells. 15 µL was taken to determine the cell density and viability using an automated cell counter (Nexcelom Cellometer Auto 2000) with the AO/PI (acridine orange / propidium iodide) staining solution (Nexcelom, CS2-0106). The live cell density was 797,000 per mL with a viability at 92.0%. The remaining cell suspension was used for single cell capture on a 10X Genomics Chromium platform. Single cell capture and library construction were performed using the 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit (v2 Chemistry) following the manufacturer's instructions. The libraries were sequenced on an Illumina NextSeq500 sequencer.