Single cell RNA sequencing of fluorescent expressing Pleurodeles waltl spleens
Single cell RNA sequencing of fluorescent expressing Pleurodeles waltl spleens
Newts were narcotized in 0.05% tricaine (MS222) in housing water. Spleens were harvested from three separate Pleurodeles waltl and processed as individual samples in parallel. All animals were post-metamophorsis but not yet sexually mature and included: one female tgTol2(CAG:Nucbow CAG:Cytbow)Simon (5.67g weight, 10.8cm snout-to-tail length), one male tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon (5.36g, 11.1cm), and female tgTol2(CAG:loxP-Cherry-loxP-H2B::YFP)Simon  (6.25g, 10.8cm).  Forceps and iridectomy scissors were used to remove the spleen in one piece, making sure that the forceps did not tear the spleen. Iridectomy scissors were used to carefully remove connective tissue.  A 70μm nylon mesh filter was inserted into a 50mL conical and 1mL of ice cold 0.7X PBS was added to pre-wet the filter. The spleen was placed on the pre-wetted filter and slowly mashed through the filter using the plunger stopper end of a 3mL syringe. Once the spleen appeared translucent, the plunger and filter were thoroughly washed with ~10mL ice cold 0.7X PBS and making sure no PBS was left on the plunger or filter. The 0.7X PBS solution was then poured into a 15mL conical and centrifuged at 300g for 5 min at 4℃ in a swinging bucket rotor. Supernatant was decanted and cells were resuspended in 1mL of 0.7X PBS. Cells were then counted using trypan blue to assess viability. Viabilities of non-erythroid cells (based on cellular morphology) were 85%, 98.4% and 91.6% in eBFP, eGFP, and mCherry animals, respectively. Fluorescent activated cell sorted was used to sort for fluorescent positive cells. Erythrocytes do not express fluorescent markers and are far larger than other splenocytes and were thus excluded using lack of fluorescent expression and size. In total, 4x10^5 cells of GFP+, 4x10^5 of mCherry+ and 3.15x10^5 BFP+ were isolated in individual 1.5mL eppendorf tubes. GFP and mCherry expression was marked, but BFP expression was dim. 500μL of each solution was then added to an individual 1.5mL eppendorf for the fluorescent pool sample. This was centrifuged in an eppendorf at 4℃ at 300g on a tabletop centrifuge. The supernatant was carefully removed and resuspended in the remaining volume.  Cells were then manually counted and adjusted to a concentration targeting the collection of 10,000 cells on the 10x Genomics controller.