GSM7290666: 5cycles, replicate 1, scRNAseq; Schmidtea mediterranea; RNA-Seq
For each biological replicate, 10 animals were dissociated into single-cell suspension by dicing in CMFB buffer [calcium-magnesium-free solution with 1% BSA (400 mg/L NaH2PO4, 800 mg/L NaCl, 1200 mg/L KCl, 800 mg/L NaHCO3, 240 mg/L glucose, 1% BSA, 15 mM HEPES, pH7.3)] and nutating for 2 hours at room temperature. The cells were centrifuged at 500g for 5 min, resuspended, and filtered through a 30µm cell strainer (BD Biosciences, cat.no 340628) to remove debris. The concentration of filtered cells was calculated with a TC20 automated cell counter (Bio-Rad). After centrifugation, cell concentration was adjusted to 100,000 cells/mL with staining buffer [CMFB containing DRAQ5 (5 µM) and Calcein-AM (0.4 µM)] and nutated at room temperature for 5 min. X1 cells were gated for vital 4N cells (DRAQ5+ Calcein-AM+) on a Sony MA900 Cell Sorter. 100,000 cells were sorted and diluted to a concentration of 1000 cells/µL for subsequent library preparation. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics) for untreated samples. For DASH-treated samples, 30 sgRNAs targeting 16S sequences were generated by in vitro transcrption. 1 ng of the cDNA was incubated with 30nM of Cas9 and 300nM of sgRNAs for overnight digestion. Digested cDNA were PCR-clean up by Ampure beads, and followed by 10 cycles of PCR using R1 and TSO primers. The subsequent library preparation for DASH-treated samples were according to manufacter's instructions for end repair and indexing.