title: Comparative RNA-seq analysis of CD146⁺ pericyte transcriptomes from wild-type and mdx mice
design: Comparative RNA-seq analysis of CD146⁺ pericyte transcriptomes from wild-type and mdx mice
library_construction_protocol: CD146+ cells from both mouse strains were isolated as previously described. Briefly, isolated muscles were cut into smaller pieces, incubated in 0.2% collagenase type I in DMEM at 37 ° C for 1.5 h, followed by the additional 30 min of incubation in dispase solution in DMEM solution (2 U/ml). Cell suspension was filtered, centrifuged, and cells expressing CD146 were selected using magnetic columns (MACS; Miltenyi Biotec) and antibodies against CD146 conjugated with ferromagnetic particles, according to the manufacturers protocol. Sorted CD146+ cells were suspended in growth medium, that is, DMEM (glucose 4.5 g/l) supplemented with 15% FBS and 1% penicillin/streptomycin, seeded in culture plates and expanded under standard conditions: 37 °C, 5% CO2. Cell were cultures for 5 days, then trypsynized and subjected to RNA isolation. Total RNA was extracted using High Pure Isolation Kit (Roche). For the total RNA-seq procedure, 275 ng of RNA (RIN>9) obtained from cultured WT or mdx CD146+ cells was purified using KAPA Magnetic Beads (KAPA Biosystems, cat no. 07983298001) and then subjected to ribodepletion and library construction using the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems, cat. no. 08098140702) according to the manufacturer's protocol. The adapters used were KAPA_Univeral adapter and KAPA UDI primer mixes (KAPA Biosystems, cat. no. 9063781001, 9329838001). The samples were fragmented: 94 ° C 5 min, 12 enrichment cycles were performed. The resulting libraries were subjected to fragment length control using an Agilent TapeStation 4150 and a High Sensitivity D100 Reagent Kit (Agilent, cat. No.5067-5585) according to the manufacturer's protocol. 