GSM9594597: Sorted Olfr2 + Cells - 2C; Mus musculus; RNA-Seq
We performed low-input bulk RNA-seq analysis on sorted aortic F4/80+ CD64+ macrophages from Apoe−/− mice categorized as Olfr2+ and Olfr2− (n = 3 per group). To achieve this, we employed the Smart-Seq2 method, which enabled us to obtain comprehensive transcriptomic profiles from a small number of sorted cells, ranging from 100 to 200. For Smart-Seq2, sorted single cells are directly lysed in a buffer containing 0.1% Triton X-100, deoxynucleotide triphosphates, and 40 U/μL RNase inhibitor in molecular-grade water. We sorted at least three technical replicates from pooled aorta cell suspensions of four female Apoe−/− mice subjected to a WD for 4 weeks. Samples were vortexed, centrifuged at 3000 g for 2 min, and stored at −80°C until further processing. To generate cDNA, we polymerase chain reaction (PCR) amplified 8 μL of lysed cells (corresponding to approximately 200 cells) using an 18-cycle PCR to obtain sufficient material for downstream procedures. The amplified cDNA was size-selected, and any remaining primers were eliminated through a magnetic beads double-stranded DNA capture using a 0.8:1 volume-to-volume ratio AMPure XP beads (Agencourt) procedure. The size-selected cDNA was quantified with a fluorometric DNA quantification assay (PicoGreen dsDNA; Thermo Fisher Scientific). A 1 ng of cDNA was used to generate barcoded Illumina sequencing libraries using a standard tagmentation protocol (Nextera XT library preparation kit, Illumina; nine cycles of PCR-based amplification using unique dual-index Illumina adaptors). Libraries were purified with a double size-selection protocol (0.5 followed by 0.8 volume bead/initial sample volumes) and quantified (PicoGreen quantification assay; Thermo Fisher Scientific), and the fragment size was measured by capillary electrophoresis (fragment analyser; Advanced Analytical Labs). Libraries were pooled, quantified by a quantitative PCR (qPCR) assay (KAPA SYBR FAST qPCR Kit; Roche Diagnostics), and loaded onto Illumina's NovaSeq 6000 system and sequenced with 50 base paired-end reads.`