title: 6N_69
organism: Mus musculus
description: Protocols: 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).