title: Illumina NovaSeq 6000 paired end sequencing; Single cell-RNA sequencing for identifying hemocyte subpopulations
design: Single cell-RNA sequencing for identifying hemocyte subpopulations
library_construction_protocol: Freshwater crayfish Pacifastacus leniusculus (adult intermolt males) were obtained from lake Erken (located in east-central Sweden near the Baltic coast (59.8 N 18.6 E). The animals were maintained in the crayfish facility at department of Organismal Biology, Uppsala University, in running tap water at 10-12°C, in a 12:12 light:dark cycle. The water was aerated with plastic tubing and ceramic oxygen stones. The animals were fed once a week. Each Hpt and APC was dissected from the crayfish, and digested into single cells by incubation in 300 μl of 0.1% collagenase (Type I), 0.1% collagenase (Type IV) in CPBS at room temperature for 20 min on a rotating plate. After collagenase treatment the tissue was gently passed 10–20 times through a Pasteur pipette and centrifuged at 800 g for 5 min to remove the collagenase solution. The pellet was washed once in 1 mL 0.15 M NaCl with 0.04% BSA by centrifugation at 800 x g for 5 min, suspended in 1 mL 0.15 M NaCl with 0.04% BSA and then filtered through a 40 mm cell strainer. Isolated cells from four animals were pooled to the HPT sample used for scRNA-seq. Sequencing libraries were prepared using Chromium Single Cell 3' reagent kit v3 (cat# 1000075/1000073/120262, 10xGenomics) according to the manufacturer' protocol (CG000183 Single Cell 3' reagent Kit User Guide, v3 chemistry, 10x Genomics).
attributes.tag: Experimental Factor: cell type
attributes.value: hematopoietic cell