title: FEW03_805_GMP
organism: Mus musculus
description: Protocols: All mice were housed in specific-pathogen-free conditions. C57B6/J and 129S2/SvPPasCrl mice were obtained from Charles River Laboratories. Nlrp3KO1293ES mice have been previously described16, and were maintained in-house on a C57B6/J background. Nlrp3fl/fl (strain ID: 12809) mice were obtained from Taconic. Rosa26ERt2Cre mice have been previously described21, and were obtained from Jackson Laboratories (strain ID: 008463) .  For BMDMs and GMPs RNA was extracted using Picopure RNA Isolation Kit (Thermofisher) according to manufacturer's instructions. For microglia, RNA was extracted using RNeasy Mini Kit (Qiagen). Residual DNA was removed using RNAse-Free DNase Set (Qiagen). RNA was assessed for quality and quantity (TapeStation, Agilent). Microglia were purified from adult brain tissue using the Neural Tissue Dissociation Kit (130-092-628, Miltenyi Biotec), Myelin Removal Beads II (130-096-433, Miltenyi Biotec) and CD11b Microglia Microbeads (130-093-636, Miltenyi Biotec) according to manufacturer's instructions.  For GMPs and BMDMs, RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA). For microglia, 3'mRNA Seq was performed using the QuantSeq 3'mRNA-Seq Library Prep Kit FWD (Lexogen).
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: collection date
attributes.value: not collected
attributes.tag: sex
attributes.value: male
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: common name
attributes.value: house mouse
attributes.tag: cell_type
attributes.value: granulocyte-monocyte progenitor
attributes.tag: genotype
attributes.value: NLRP3_ko
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected