title: Single cell RNA seq of Cell Multiplexing Oligo, pooled Pleurodeles waltl and Notophthalmus viridescens spleens
design: Single cell RNA seq of Cell Multiplexing Oligo, pooled Pleurodeles waltl and Notophthalmus viridescens spleens
library_construction_protocol: Newts were narcotized in 0.05% tricaine (MS222) in housing water. Spleens were harvested from two separate Pleurodeles waltl and processed as individual samples in parallel and from two Notophthalmus viridescens which were pooled as one sample. These three samples were then . All animals were sexually mature adults and included: one female Notophthalmus viridescens (4.45g and 10.6cm snout-to-tail) and one male Notophthalmus viridescens (3.55g and 10.2cm) (as a pooled sample), and one Pleurodeles waltl tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon  female (23.5g and 16.1cm snout-to-tail length), one male Pleurodeles waltl tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon  (13.95g and 15.7cm).    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 in a swinging bucket rotor. Supernatant was decanted and Notophthalmus cells were resuspended in 1mL of 0.7X PBS with 0.04% ultrapure BSA.   For Pleurodeles spleens only:  After the spleen was thoroughly mashed through the pre-wetted 70μm nylon filter and the filter was washed with 10mL of 0.7X PBS the cells were centrifuged at 300g for 5 min. Splenocytes were resuspended cells in 1mL of sterile filtered 1X ACK (http://cshprotocols.cshlp.org/content/2014/11/pdb.rec083295.short) to lyse red blood cells. After one minute of lysis, cells were diluted with 10mL of 0.7X PBS and filtered through a 70μm nylon mesh filter and centrifuged at 300g for 5 min. Cells were then resuspended 0.7X PBS with 0.04% ultrapure BSA.  Once single cell suspensions were obtained cells these three samples were stained with Cellplex reagents from 10x Genomics. The pooled Notophthalmus sample and the individual Pleurodeles samples were then taken through the 10x Genomics 3' Cellplex labeling protocol (Demonstrated Protocol, CG000391) the only modifications being the use of 0.7X PBS + 0.04% BSA for all wash and resuspension steps. Samples were stained with CM304 (Pleurodeles female), CMO305 (Pleurodeles male), and CMO306 (pool of Notophthalmus samples). Cells were then counted using trypan blue to assess viability.       Cells were then manually counted and adjusted to a concentration targeting the collection of 9,000 cells on the 10x Genomics Chromium controller.