GSM9324581: Output mouse allCell_1A; Mus musculus; RNA-Seq
For the brain experiment, electroporated brains were harvested and dissected in ice-cold sterile PBS. Meninges were removed and GFP+ portions of the cortices were incubated at 37◦C for 10 minutes in 0.25% trypsin-EDTA supplemented with 0.1% DNAse I. Following incubation, the trypsin solution was removed and replaced with ice- cold 10% FBS/HBSS/Propidium iodide supplemented with 0.01% DNAse I. A single cell suspension was then generated by trituration with a fire-polished glass pipette and filtered with a 30 μm cell strainer. For the heart experiment, at the end of the incubation period, we detached the cardiomyocytes with Accutase to create a single cell suspension. For both the brain experiment and the heart experiment, cells were then stained with the LIVE/DEAD Near-IR Dead Cell Stain per manufacturer's instructions. Following staining, viable GFP+ cells were bulk sorted using a FACS Aria II cytometer. Output libraries were constructed according to the manufacter's instructions (Chromium Next GEM Single Cell 3' Reagent Kit v3.1 protocol, 10x Genomics). Briefly, Gel bead-Cell complexess 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 Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Input plasmid libraries were fragmented and sequenced with the NEBNext Ultra II FS DNA Library Prep Kit per manufacturer's instrutions.
GEO
GSM9324581_r1