GSM7006877: intact-TAS; Dugesia japonica; RNA-Seq
Planarian was placed on a filter paper on ice and cut into small pieces using a surgical knife. The cell suspension was incubated in 1/7 × PBS(-) (NACALAI TESQUE, INC, Kyoto, Japan) containing 25 µg/mL Liberase TL (Merck, Darmstadt, Germany) or 0.25% (v/w) Trypsin (NACALAI TESQUE, INC, Kyoto, Japan) at 22℃ for 5 min. After the incubation, the cell suspension was gently pipetted at least 60 times to completely dissociate it into single cells. In order to wash out the enzymes, the dissociated cell solution was centrifuged at 200 x g for 10 minutes, followed by discarding the supernatant and re-suspension with 500 μL of 1/7 × PBS(-). After another round of the wash step, the cells were re-suspended in 1 mL of 1/7 × PBS(-) containing 1 µg/mL of Calcein AM (DOJINDO LABORATORIES, Kumamoto, Japan) and 1 µg/mL PI (DOJINDO LABORATORIES, Kumamoto, Japan), and were passed through pluriStrainer-Mini 40 μm, 20 μm, and 10 μm (pluriselect, Leipzig, Germany) to remove debris or cell aggregations. The numbers of living and dead cells were counted with a hemocytometer and fluorescence microscope BX53 (Olympus, Tokyo, Japan). The dissociated cells of D. japonica (Chiba, Japan) were subjected to a BD Rhapsody system with BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit (BD Biosciences, New Jersey, USA) following the manufacturer's instructions except for using 1/7 × PBS(-) instead of Sample Buffer. The reverse-transcribed, Exonuclease I-treated BD Rhapsody beads were subjected to TAS-Seq workflow according to the protocols described previously (Shichino et al. 2022) by ImmunoGeneTeqs, Inc (Chiba, Japan).