GSM8859512: FoxA(RNAi), rep1, scRNA-seq; Schmidtea mediterranea; RNA-Seq
For each biological replicate, tunks of 50 animals from each condition were dissected. The tissues 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 rocking for 2 hours at room temperature. The cells were washed by centrifugation 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. Nucleated Calcein-AM+ cells were collected 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) with DASH treatment. The cDNA prior to fragmentation was treated with CRISPR, which 30 sgRNAs targeting 16S ribosomal sequences. 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 were according to manufacter's instructions for end repair and indexing.