Cells were lysed with RLTPlus buffer and stored at −80 °C until RNA isolation. Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen), and mRNA was transcribed into cDNA using First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Gene expression was analyzed via qPCR and the Blue S’Green qPCR Kit (Biozym) and normalized to the expression of Rpl4. Raw data were analyzed with the BioRad CFX Maestro V 2.3 software. For human iPSC-derived microglia, we lysed the cells in LB1 buffer and isolated RNA using the NucleoSpin Kit (Macherey Nagel). Afterward, mRNA was transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Gene expression was analyzed via qPCR and the SYBR Master Mix (Applied Biosystems) and normalized to the expression of RPL0. To compensate for inter-assay variability, relative expression levels were calculated as a ratio of CD83ΔMG to CD83wtCre, or as fold change in relation to unstimulated cells. All primers were designed and validated according to the MIQE guidelines (see Table 1).Table 1Primers used for qPCRGeneOrientationSequenceApoeForward5’-TCAGCTCGAGTGGCAAAG-3’Reverse5’-TTACTTCCGTCATAGTGTCCTC-3’Ccl2Forward5’-GAGAGCTACAAGAGGATCACC-3’Reverse5’-GATCTCATTTGGTTCCGATCC-3’Ccl3Forward5’-TCTGTCACCTGCTCAACATC-3’Reverse5’-CGATGAATTGGCGTGGAATC-3’Ccl4Forward5’-GCTGTTTCTCTTACACCTCCC-3’Reverse5’-GTTCAACTCCAAGTCACTCATG-3’Ccl5Forward5’-CCCACGTCAAGGAGTATTTCT-3’Reverse5’-ACCCTCTATCCTAGCTCATCTC-3’Cd83Forward5’-CGCAGCTCTCCTATGCAGTG-3’Reverse5’-GTGTTTTGGATCGTCAGGGAATA-3’Clec7aForward5’-GAATCCTGTGCTTTGTGGTAG-3’Reverse5’-GGAGCCACCTTCTCATCTAAAG-3’Entpd1Forward5’-ACCGAACTGATACCAACATCC-3’Reverse5’-CCTTCCTCTTGTCCAGTGATG-3’IfngForward5’-GCTTTGCAGCTCTTCCTCAT-3’Reverse5’-GTCACCATCCTTTTGCCAGT-3’Il6Forward5’-ACAAAGCCAGAGTCCTTCAGAG-3’Reverse5’-GAGCATTGGAAATTGGGGTAGG-3’Il12aForward5’-AACAGGGTGATGGGCTATC-3’Reverse5’-TGAGATGTGATGGGAGAACAG-3’Il23aForward5’-GCAACTTCACACCTCCCTAC-3’Reverse5’-CGAAGGATCTTGGAACGGAG-3’LplForward5’-TAGACAACGTCCACCTCTTAG-3’Reverse5’-ACATCTACAAAATCAGCGTCATC-3’Mmp9Forward5’-GCTGACTACGATAAGGACGGCA-3’Reverse5’-TAGTGGTGCAGGCAGAGTAGGA-3’Msr1Forward5’-AGGTGTTAAAGGTGATCGGG-3’Reverse5’-ATCTTGATCCGCCTACACTC-3’Rpl4Forward5’-GCTGAACCCTTACGCCAAGA-3’Reverse5’-TCTCGGATTTGGTTGCCAGT-3’SiglechForward5’-GCAAGAGATCCCATACGAAGAG-3’Reverse5’-AGTCCAGTTGGCACCATC-3’Spp1Forward5’-ACTTTCACTCCAATCGTCCC-3’Reverse5’-GTCCTCATCTGTGGCATCAG-3’Tbx21Forward5’-AGCAAGGACGGCGAATGTT-3’Reverse5’-GGGTGGACATATAAGCGGTTC-3’Tmem119Forward5’-GTGTCTAACAGGCCCCAGAA-3’Reverse5’-AGCCACGTGGTATCAAGGAG-3’TnfForward5’-GTGATCGGTCCCCAAAGGG-3’Reverse5’-CCAGCTGCTCCTCCACTTG-3’Trem2Forward5’-GGAACCGTCACCATCACTC-3’Reverse5’-CCAGCATCTTGGTCATCTAGAG-3’CD83 (human)Forward5’-TGCTGCTGGCTCTGGTTATT-3’Reverse5’-TGTGAGGAGTCACTAGCCCT-3’RPLP0 (human)Forward5’GCCATTGCCCCATGTGAAGT3’Reverse5’AGCTGCACATCACTCAGGATT3’
