GSM9228750: LDLR_KO; Mus musculus; RNA-Seq
Aortic arches were dissected and maintained in HypoThermosol® FRS Preservation Solution with transcription inhibitor flavopiridol on ice. Tissues were minced and digested at 37 °C for 15 minutes in an enzyme cocktail containing 1 mg/ml collagenase I, 1 mg/ml dispase, 150 mg/ml DNase I, and Liberase™ TL. Dissociated cells were filtered through a 100 µm strainer, stained with live/dead dye, and subjected to FACS sorting. Live singlets were collected for single-cell RNA sequencing FACS-sorted single-cell suspensions of aortic arches from iLpl-/-/Ldlr-/- and Lplfl/fl/Ldlr-/- mice were used to generate barcoded single-cell cDNA libraries using the PIPseq™ V3 T20 Single Cell RNA Kit (Fluent Biosciences), which profiles up to 20,000 cells per reaction. Sequencing libraries were constructed from one pooled sample of four mouse aortic arch samples across four libraries and combined for downstream processing. Sample multiplexing was performed with TotalSeq™-A Hashtag Antibodies (BioLegend; A0301, A0302, A0303, A0304) for cell hashing. Libraries were loaded onto an Illumina NextSeq-600 platform for sequencing.