GSM8280221: Posterior1; Schmidtea mediterranea; RNA-Seq
Tissue sections from 15-20 planarians were finely diced into small fragments (<1 mm) and transferred to a 15 ml conical with CMFB (Calcium-Magnesium Free solution; 400 mg/L NaH2PO4, 800 mg/L NaCl, 1200 mg/L KCl, 800 mg/L NaHCO3, 1.33 mM glucose, 15 mM HEPES pH 7.3, 1% BSA) supplemented with 10 μg/ml collagenase. Cell dissociations were assisted by mechanical agitation on a nutator with intermittent pipetting. Cells were dissociated for ~10 minutes, passed through a 40-μm filter, and pelleted by centrifugation for 5 minutes at 310 x g. After supernatant removal, the cell pellet was resuspended in 4 ml of CMFB. Cells were incubated in Hoechst 33342 (10 mg/mL) for 45 minutes and then stained with propidium iodide (PI; 10 µg/µL) immediately before sorting on a FACSAria (BD Biosciences). Library was performed according to the manufacturer's instructions - 10X Genomics 3' Single Cell RNA-Seq v3.1 protocol