GSM9472809: Progeria_10weeks_batch3_P326_not_GFP_sorted_Protocol1; Mus musculus; RNA-Seq
Dissected aortic arches were processed to generate single-cell suspensions using enzymatic digestion. Two digestion protocols were applied. For both protocols, the aortic arches were first opened longitudinally and cut into approximately 1 mm fragments before enzymatic treatment.  In protocol 1, individual aortic arches were digested in HBSS (Gibco, 14025-092) supplemented with 2% bovine serum albumin (Merck, A9647), collagenase I (250 µg/ml; Merck, C5894), collagenase II (250 µg/ml; Merck, C6885), collagenase IV (250 µg/ml; Merck, C1889), porcine elastase (5.98 U/ml; Worthington Biochemical, LS002279), 2.5 mM CaCl₂, and DNase (125.75 U/µl; Invitrogen, 18047-019). Samples underwent three sequential digestion steps of 10 minutes each at 37°C with shaking at 450 rpm, with 20 resuspensions performed between each incubation. Following digestion, cell suspensions were passed sequentially through 70 µm and 40 µm strainers to remove remaining tissue fragments.  In protocol 2, aortic arches were digested in 24-well culture plates using sterile DMEM supplemented with collagenase IV (2.5 mg/ml; Gibco, 17104-019) and porcine elastase (2.5 U/ml). Digestions were carried out for 2 hours in a humidified incubator at 37°C and 5% CO₂, with gentle resuspension every 30 minutes. Digestion was terminated by transferring the cell suspension to a 50 ml Falcon tube containing 1% BSA in PBS. The resulting suspension was then filtered through 70 µm and 40 µm cell strainers.  After digestion, the arterial cells were pelleted by centrifugation for 5 minutes at 300 × g and 8°C, then resuspended in 1% BSA in PBS. Cell viability was evaluated by staining with 5 µM DRAQ5 (Thermo Scientific, #62251) and 9 µg/ml Calcein Green (Invitrogen, C34852). Following a 15-minute incubation on ice, individual arterial cells were sorted using a BD FACS Aria Fusion based on size and double-positivity for Calcein Green and DRAQ5 into 384-well plates containing Smart-seq2 lysis buffer. In selected experiments, GFP-based sorting was performed to enrich for VSMCs (Table 1). Immediately after sorting, plates were sealed, frozen on dry ice, and stored at −80°C until further processing. Sequencing was performed at the Single Cell Core Facility of Flemingsberg (SICOF) using  Smart-seq2. Briefly, SuperScript II reverse transcriptase and oligo(dT) primers (ThermoFisher Scientific) were used for cDNA synthesis, with template-switching oligonucleotide for second-strand cDNA synthesis. 23–26 PCR cycles were done for cDNA amplification. TapeStation 4200 or a 2100 Bioanalyzer with a DNA High Sensitivity chip (Agilent Biotechnologies) were used for  quality control (QC) measurements. Samples that passed QC were subsequently fragmented and tagmented) using the Tn5 transposase, and each well was uniquely indexed using the Illumina Nextera XT index kits (Sets A–D). Finally, all uniquely indexed cDNA libraries from a 384-well plate were pooled into a single sample and sequenced on one lane of an Illumina HiSeq 3000 instrument using dual indexing and single-end 50 base-pair reads