GSM6247961: late9_05_C10; Ambystoma mexicanum; RNA-Seq
Blastemas samples were collected from mid- (13 dpa), late (15 dpa)- or palette-stage (18 dpa) animals at 48 h following GalNP(Rho) injection. Samples were dissociated in 400 ml of 1x Liberase (Roche) diluted in 0.8x PBS, using mechanical disruption with forceps together with smooth pipetting at room temperature for 30 min. For live/dead staining, suspensions were incubated in 0.8X PBS supplemented with 200 nM CellTrace calcein green AM (Invitrogen) and 12.5 uM SYTOX blue (Invitrogen) at room temperature for 15 min. Subsequently, cell suspensions were filtered through a 70-mm-diameter strainer to generate single-cell suspensions and immediately subjected to FACS. Alive cells were sorted using a FACSAria III equipped with a 100 mm nozzle (BD Biosciences) according to the presence or absence of rhodamine signal using blastemas from vehicle-injected axolotls as negative controls. Single rhodamine+ cells were sorted into 2 μl of lysis buffer (nuclease-free water, 0.2% Triton X100, 4U/μl RNase Inhibitor). Processing of the samples for RNA-sequencing was done following a modified Smartseq-2 protocol (https://star-protocols.cell.com/protocols/1595). In brief, samples were thawed on ice and we added 0.5ul of dT-buffer to each sample, containing poly-dT oligos (final concentration of 0.5uM) for capturing mRNAs. Subsequently, we performed reverse transcription and cDNA amplification. Libraries were prepared with the Illumina® DNA Prep Tagmentation kit and sequenced on an Illumina NovaSeq 6000 S1 flow cell to an average of 0.85 mio PE50bp fragments/cell.