Ten planarian tail fragments were transferred to a clean 35 mm Petri dish in ~1 mL planaria water. The water was replaced with 500 uL Calcium/Magnesium Free Buffer (CMFB: 15mM Hepes, 400mg/L NaH 2 PO4 , 800mg/L NaCl, 1200mg/L KCl, 800mg/L NaHCO3 , 240mg/L glucose, 1% BSA, pH7.3). Tissue was minced by repeatedly cutting with a razor blade, washed to a conical tube, and mechanically disrupted by forceful pipetting. Dissociated cells were pelleted by centrifugation, resuspended, and filtered through a 70 micron cell strainer. Single cell libraries were constructed from live, single cell suspensions according to the manufacturer's directions using the Chromium iX single cell instrument and Chromium GEM-X Single Cell 3' Reagent kit v4 (10x Genomics), capturing ~13-15K cells per sample and sequencing to a minimum depth of 20,000 reads/cell on a Singular Genomics G4 instrument.