GSM9067353: Head regenerating fragment, 72hpa; Hydra vulgaris; RNA-Seq
Samples were incubated in Pronase E solution (7.5 mg/mL of pronase E diluted in dissociation medium [6 mM CaCI2, 1.2 mM MgSO4, 3.6 mM KCl, 12.5 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 6 mM sodium pyruvate, 6 mM sodium citrate, 4 mM glucose; pH 6.9]) for 1h at 24°C, 300rpm, protected from light. Animals were dissociated into single cells mechanically by pipetting up and down. Cells were strained through a sterile CellTrics® 30µm cell strainer, collected in a centrifuge tube and spin 5min, 300xg at room temperature (RT). Supernatant was removed and cells were resuspended in dissociation medium + 0.01% Bovin serum albumin (BSA). Cells were centrifuged again and resuspended in dissociation medium + 0.01% BSA. Cells were strained and collected in a FACS tube. Dead cells were stained with DRAQ7 and excluded from the FACS sort. Cells were collected in a PBS + 0.04% BSA, without Cacl2 and MgCl2, solution. FACS-sorted cells (16K) were immediately processed for scRNA-seq using 10X genomics Chromium Single Cell 3' Reagents v3.1 with UDI indexes, according to the manufacturer's protocol.