Effects of mouse genetic background on neutrophil gene expression in autoinflammatory osteomyelitis
Effects of mouse genetic background on neutrophil gene expression in autoinflammatory osteomyelitis
Mice were sacrificed by Cervical dislocation. Hind paw tissue was cut into small pieces and crushed using mortar and pestle in PBS with 2% FCS. Cell suspension was centrifuged (500 x g, 5 min, 2°C) and pellets were reconstituted in ACK buffer (150 mM NH4Cl, 0.1 mM EDTA (disodium salt), 1 mM KHCO3). Cell suspension was centrifuged and pellet was reconstituted in PBS following neutrophil isolation using mouse Neutrophil Isolation Kit (Miltenyi Biotec, catalog # 130-097-658) and autoMACS Pro magnetic cell separator (Miltenyi Biotec). Suspension of purified neutrophils was centrifuged and pellet was reconstituted in lysis buffer from Rneasy Micro Kit Qiagen, cat#74004, following RNA isolation according to manufacturer's instructions of this kit. RNA from purified neutrophils was isolated using Rneasy Micro Kit Qiagen, cat#74004), mRNA was treated with Dnase I (Qiagen, cat#1023460) for 15 min. Libraries were prepared with a SMARTer® Stranded Total RNA-Seq – Pico Input Mammalian library preparation kit v2 (Takara).