GSM6246469: Mid bud Rho-, replicate 01, bulk RNA seq; Ambystoma mexicanum; RNA-Seq
Blastemas samples were collected from early (11 dpa), 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 ?l of lysis buffer (nuclease-free water, 0.2% Triton X100, 4U/?l 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 Vazyme TruePrep DNA Library Prep Kit V2 (discontinued) and sequenced on an Illumina NextSeq 500 to an average of 32mio SE75bp fragments/sample.