GSM8967697: Blood cells, biological replicate 2; Ciona robusta; RNA-Seq
Ciona robusta adults were collected from the wild in Carlsbad, California, USA by M-REP. They arrived in the lab <24 hours after collection, where they were kept at 17–18ºC in 35–40 ppt artificial seawater (Instant Ocean SS15-10) and fed 6–7 days a week with 15 mL of Phyto-Feast (Reef Nutrition). Blood was collected 7 days after arrival in the lab, during which time animals remained healthy by visual inspection. To reduce movement during dissection, animals were relaxed by incubating 3–5 adults for 3–4 hours at 4 °C in 400 mL artificial seawater from the aquarium system with 5 g of 99% L-menthol crystals (Thermo Scientific A10474). Volume and weight measurements were approximate. To collect blood, animals were dissected to expose a large blood vessel adjacent to the heart and running along the endostyle (“ventral vessel” in Millar 1953). Blood was drawn from the vessel with a zero dead space tuberculin syringe with a 25G needle (Exelint 26046), then transferred to a microcentrifuge tube on ice. This tube had first been coated with bovine serum albumin (BSA) by washing with 0.5 mL of 10% (v./v.) BSA (Sigma 9048-46-8) in Dulbecco's phosphate-buffered saline (DPBS, Gibco 14200075). Blood was harvested from 7. Equal blood volumes were then pooled for animals A, B, C, D, and then again for animals A, E, F, G, such animal A contributed blood to both pools. Blood was then filtered through a 40 µm strainer (pluriSelect 43-10040-60) and transferred to a microcentrifuge tube, which had been coated with bovine serum albumin (BSA) by washing with 0.5 mL of 10% (v./v.) BSA (Sigma 9048-46-8) in Dulbecco's phosphate-buffered saline (DPBS, Gibco 14200075). Blood cells were pelleted by spinning at 800 g for 10 minutes at 4 °C in a swinging bucket centrifuge (Eppendorf Centrifuge 5810R). The supernatant was removed, and cells were resuspended in 0.7 M D-mannitol (Sigma Aldrich M4125) in 1X PBS (gibco 70011-044) (PBS-M). This PBS-M buffer was optimized for scRNA-seq of cells adapted to high salinity environments. Cells were then filtered through a 40 µm strainer once more to remove large clumps. Library preparation was done with Chromium Next GEM Single Cell 3′ v3.1 Kits (10X Genomics 1000123, 1000127, and 1000190). We followed the manufacturer's guidelines except for the preparation of the RT mix prior to cell encapsulation. The manufacturer's protocol directs users to add nuclease-free water, followed by cell suspension; instead of adding nuclease-free water, we added an equivalent volume of PBS-M.