GSM8310812: SIGAA2. replicate 1, scRNAseq; Xenopus laevis; RNA-Seq
Ectodermal explants were isolated from IVF and NT embryos of stage 12 and shortly washed in Newport 2.0 dissociation buffer (100 mM sodium isethionate, 20 mM sodium pyrophosphate, 10 mM CAPS, 20 mM glucose, pH10.5 adjusted by NaOH) to remove calcium ions. Ectodermal explants were transferred into fresh dissociation buffer in BSA coated low bind microcentrifuge tubes and incubated for 30min at 18°C with agitation. During the period of incubation, tubes were inverted several times. Resulting single cell suspensions were resuspended in PBS-BSA solution and filtered through 30 µm cell strainers. Cells were washed 3-times with 1 ml PBS-BSA, counted and analyzed for viability. 2500 cells with viability more than 95% and not detectable RNA in supernatant were submitted for library preparation. Libraries were constructed according to the manufacturer's instructions (single-cell 3' v2 and v3 protocols, 10x Genomics). GCs were resuspendend in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R! primer sequence, Unique Molecular Identifier (UMI) and the 10x barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.