GSM5651893: ACE0_1p2h-2; Mus musculus; RNA-Seq
The main olfactory epithelium was dissected and cleaned up in the Earle's Balanced Salt Solution (EBSS, pre-treated with carbogen for at least 5 minutes before each use, Worthington), then transferred to a round-bottomed glass dish containing 750 µL of papain solution (one vial of Papain, (Worthington) dissolved in 5 mL of EBSS and then equilibrated 10 minutes at 37 °C) and 100 µL of DNase solution (one vial of DNase-I (Worthington) dissolved in 500 µL of EBSS). Bone was removed from the epithelium under a dissecting microscope and the resultant tissue was placed in a 5 mL tube (Becton Dickinson) with an additional 1.75 mL of Papain solution and 200 µL of DNase-I solution and rocked gently for ~60 minutes at 37 °C. The tissue was then gently triturated with a 5 mL pipette 10-15 times, passed through a 40 µm cell strainer (Becton Dickinson), and washed with 1 mL of Hibernate-A medium containing 10 % Fetal Bovine Serum (FBS, GIBCO). Filtered cells were transferred to a new 5 mL tube and centrifuged 5 minutes at 300 x g. The supernatant was decanted, the cells were washed once with 4 mL Hibernate-A containing 10 % FBS and resuspended in 1 mL Hibernate-A containing 0.2 % FBS. Each of the solutions above contained transcriptional inhibitors (5 µg/mL of Actinomycin D, 10 µg/mL of Anisomycin and 10 µM of Triptolide, all obtained from Sigma) for all experiments, except where noted (Table S1). Dissociated cells were stained with propidium iodide (final concentration 1.65 µg/mL), and subject to FACS to remove dead cells and obvious doublets. For GFP-expressing mouse lines (OMP-IRES-GFP, P2(P2 locus), M72(S50 locus) and S50(M72 locus)), GFP positive and negative cells were sorted separately using a FITC filter. Cells were sorted into Hibernate-A containing 5 % FBS (also include 0.1x of transcriptional inhibitors), centrifuged for 5 minutes at 300 g, and resuspended with PBS. Prepared single cell suspensions were used to prepare single cell RNA-seq library with the chromium single cell gene expression system (chromium single cell 3' reagents and GEM v2, v3 or v3.1 dual index, 10x genomics), using the default protocols provided by 10x genomics. Each of the replicates other than GFP-positive ones was aimed to have 10,000 cells. The expected multiplet rate is ~8.0 %. single cell 3' end RNA-seq