GSM8324587: Aorta from Ldlr-/-AAV-Gls2shRNA; Mus musculus; RNA-Seq
Whole aortas from 3 to 4 animals were flushed, dissociated with a scalpel and dounce homogenization was performed in 1mL chilled lysis buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 1mM DTT, 0.4U/mL RNase inhibitor (LifeTechnologies-N8080119) and 0.4U/mL Riboblock (FisherScientific-EO0381)) according to 10X Genomics protocol. Single nuclei suspension was recovevered after centrifugation (500 g for 5min at 4°C) in 250μl of chilled lysis buffer and mixed 1:1 with density gradient medium (40mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl pH7.4 in OptiPrep (Sigma-D1556)). Enrichment of nuclei fraction was achieved by cushioned-density gradient centrifugation with a 29% Optiprep density gradient medium (129mM sucrose, 77.5mM KCl, 15.5mM MgCl2, 31mM Tris-HCl pH7.4 in OptiPrep (Sigma-D1556)) and centrifugation at 13,500 g for 20min at 4°C. Nuclei pellet was resuspended in 30μl wash buffer (PBS containing 1% BSA and 0.4U/mL RNase inhibitors) and directly processed to 10x Genomics protocol Library was performed according to the manufacter's instructions (chromium Single-Cell 3' v3.1 kit, 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.