GSM9790680: WT_SPM_rep4; Mus musculus; RNA-Seq
Peritoneal lavage cells were isolated by injecting 10 ml PBS containing EDTA into the peritoneal cavity of euthanized mice and collecting the fluid with a syringe. Bone marrow cells were obtained by dissecting the hind limbs, removing the muscle, cutting the femur and tibia bones and flushing with 1 ml PBS containing EDTA. Lungs were removed and chopped finely, before digestion with 0.8 mg/mL Dispase II, 0.2 mg/mL Collagenase P, and 0.1 mg/mL DNase I (Roche) in HBSS shaken at 800 rpm, at 37°C for 45 min. Lungs were filtered through a 40 µm EASYstrainer mesh (Greiner Bio-One), and RBC were lysed. FACS sorted monocytes from the bone marrow, MHCII negative macrophages from the lungs, and SPMs from the peritoneal cavity of WT and ACKR2-/- mice were isolated. RNA extraction was performed on sorted samples for RNA sequencing. RNA was extracted using the RNeasy Microkit with on-column DNAse I digestion (Qiagen), following manufacturer's instructions. 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 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.