GSM6123616: LGE, WT, 8 samples; Mus musculus; RNA-Seq
Embryonic mouse (E14.5) brains were quickly collected and placed in HBSS. The LGE was dissected and incubated in 1 mg/ml papain in HBSS for 20 min at 37 °C. The tissues were gently dissociated into a single-cell suspension by pipetting. Cells were centrifuged and washed twice, filtered through Flowmi Tip 40 μM strainers, and resuspended in HBSS+ 0.04% BSA. Cell viability was assessed by trypan blue exclusion, and the cell concentration was adjusted for targeted sequencing of 10,000 cells/sample using a Chromium droplet-based sequencing platform (10X Genomics) Single Cell 30 Reagent Kits v3 protocol to prepare the libraries following the manufacturer's instructions. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs were resuspended 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 retrotranscripted to cDNA, which contains an Ilumina R1 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 apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.