GSM9471713: Vehicle_rep1; Mus musculus; RNA-Seq
Excised inguinal fat pads were digested for 1 hour using Liberase (0.44 Wunch units/sample).  Macrophages were magnetically isolated from digested tissue using Anti-F480 MicroBeads Ultrapure (Miltenyi Biotech). Purity of the samples were tested using flow cytometry and RNA extraction was performed on samples with >90% F480+CD64+ cells. After F480-magnetic positive selection, the eluted cell suspensions from 6 inguinal fat pads were pooled together to obtain enough ATMs (around 30,000 cells) for low-input RNA sequencing. Cells were spun down at 1000g for 10 minutes, lysed, and spun again to remove bound magnetic beads before proceeding with RNA extraction. RNA was extracted using the RNeasy Microkit with on-column DNAse I digestion (Qiagen), following manufacturer's instructions, obtaining samples of around 100-150ng of total RNA in water.  RNA samples were then sent to a commercial sequencing provider (Novogene), for RNA QC, mRNA library preparation (poly-A enrichment) and Illumina Sequencing Total RNA was amplified using  SMARTer Ultra Low Input RNA Kit for Sequencing (Takara Bio) and reverse transcribed into the first-strand cDNA. The double-strand cDNA was synthesized by LD-PCR amplification, followed by the purification with AMPure XP beads and quantification with Qubit. The cDNA samples were then fragmented, end-repaired, A-tailed, and ligated with adaptors. After size selection and PCR enrichment, the RNA library was ready for sequencing.