GSM8733430: Macrophage, Mafb KO Male, rep2; Mus musculus; RNA-Seq
Two hundred fifty thousand bone marrow monocytes from Mafbfl/fl or Lyz2CreMafbfl/fl mice were isolated using Monocyte Isolation Kit (BM) (Miltenyi Biotec, 130100629) and were seeded in tissue culture-treated 6-well plates (Greiner, 657160) and cultured for 4 days in RPMI supplemented with 10% FBS, 1 mM Sodium Pyruvate (Gibco, 11360070), 1X MEM Non-Essential Amino Acids Solution (Gibco, 11140050), 50 µM β-mercaptoethanol (Gibco, 31350010) and 40 ng/mL CSF-1 (Peprotech, 315-02) to differentiate them into bone marrow monocyte derived macrophages (BMDM). After 4 days of differentiation, non-adherent cells were washed off with ice-cold PBS and total RNA was isolated with the standard TRIzol (Invitrogen, 15596018) RNA extraction protocol. Total RNA was further purified and concentrated using a RNA Clean & Concentrator Kit (Zymo Research, R1013) according to manufacturer's instructions. RNA quality and quantity were evaluated using a 5200 Fragment Analyzer (Agilent). Samples with a RIN > 9.9 were selected for sequencing. One hundred nanograms of RNA was used to generate the libraries using the TruSeq Stranded mRNA kit (Illumina, 20020594). These libraries were sequenced on an NovaSeq 6000 (Illumina) sequencer on an S4 flow cell.