GSM8758369: VEDS Fludro, rep3; Mus musculus; RNA-Seq
Between 10am and 1pm, the aorta was perfused with 10mL ice cold 1x DPBS (ThermoFisher Scientific, J67670-K2) through the right ventricle of the heart. The descending thoracic aorta (proximal to the left subclavian down to the diaphragm) was collected and the adventitia was stripped from the aortas using forceps. The aortas were then flash frozen in an empty cryovial and stored in liquid nitrogen at -154°C until processing. Within a treatment group (N = 3), aortas were randomized to determine processing order. Aortas were processed in batches containing one aorta per treatment group (total aortas per batch N = 6). Within a batch, aortas were randomized to minimize processing order bias. For each batch, the first 3 aortas were processed on wet ice by cutting the aorta into pieces in a cross hatched manner. These pieces were put into the lysis buffer from the Chromium Nuclear Isolation Kit (10x Genomics). Each aorta was manually homogenized with the supplied pestle by twisting 10 times then releasing pressure, this was repeated 4 times. Then the Chromium Nuclear Isolation Kit was used according to the manufacturer's instructions until the samples were in the 1mL Wash and Resuspension Buffer containing RNase inhibitor. At this point, the 3 processed aortas remained on wet ice and the remaining 3 samples (which had remained frozen on dry ice) were processed just like the first 3 aortas. Once all 6 aortas were in the Wash and Resuspension Buffer containing RNase inhibitor, all 6 samples were spun down and resuspended in 50uL Wash and Resuspension Buffer. Then samples were submitted on wet ice to the Johns Hopkins Single Cell & Transcriptomics core for QC and cDNA synthesis. Briefly, nuclei were combined with RT reagents and loaded onto 10X Next GEM Chip M along with 3' HT gel beads. The NextGEM protocol was run on the 10X Chromium X to create GEMs which are broken using Recovery Agent and cDNA is cleaned, following manufacturer's instructions using MyOne SILANE beads. Fragmentation, end repair, and A-tailing are completed, and samples are cleaned up using double sided size selection (0.6X, 0.8X) with SPRIselect beads. Libraries are sequenced on the Illumina NovaSeq 6000 using v1.5 kits, targeting 50K reads/cell, at read lengths of 28 (R1), 10 (i7), 10 (i5), 91 (R2).