title: GSM8869531: Blood cells, mannitol buffer; Ciona robusta; RNA-Seq
library_construction_protocol: Animals were relaxed by incubating for 3 hours at 4 °C in about 400 mL artificial seawater with approximately 5 g of 99% L-menthol crystals (3 adults per 400 mL volume). Relaxed adults were dissected to expose the endostyle and large blood vessel on the ventral side of the animal. Blood was drawn from this vessel using a zero dead space tuberculin syringe with a 25G needle, then transferred to a microcentrifuge tube on ice which had previously been coated with bovine serum albumin (BSA) by washing with 0.5 mL of 10% (v./v.) BSA in Dulbecco's phosphate-buffered saline (DPBS). Pooled blood was split into two tubes coated with 10% (v./v.) BSA in DPBS. Cells were pelleted by spinning at 800 g for 10 min at 4 °C in a swinging bucket centrifuge. The supernatant was gently removed, and cells were resuspended to a concentration of 1000-2000 cells/uL in the appropriate buffer: Ca2+- and Mg2+-free artificial seawater (CMF-ASW; 0.5 M NaCl, 9 mM KCl, 5 mM HEPES, pH 7.4) for Cr_blood_asw, or 0.7 M D-mannitol in PBS for Cr_blood_mannitol. Library preparation was performed following the manufacturer's instructions for Chromium Next GEM Single Cell 3′ v3.1 Kits (10X Genomics 1000123, 1000127, and 1000190), with the following exception: When preparing the RT mix prior to cell encapsulation, the manufacturer protocol instructs to add nuclease-free water, then to add the cell suspension. The Cr_blood_asw sample was diluted with nuclease-free water as directed. The Cr_blood_mannitol sample was instead diluted with 0.7 M D-mannitol in PBS.