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. Hemocytes were collected by bleeding  2 mL in a 1:1 volume with anti-coagulant solution (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM EDTA, pH 4.6) using a 18 G needle (BD microlane). Samples were centrifuged for 5 min at 800 × g. The supernatant was discarded, and the cell pellet washed in 2 mL 0.15 M NaCl. The wash-ing was repeated two more times, and finally the hemocytes were suspended in 1 mL 0.15 M NaCl with 0.04% BSA and used as hemocyte sample for scRNA-seq. The cell numbers in both samples were determined with a hemocytometer. 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: hemocyte