Illumina NovaSeq 6000 paired end sequencing; Cell-type-specific regulation of IL-1-driven inflammation in fungal infection
Cell-type-specific regulation of IL-1-driven inflammation in fungal infection
Mice were bred and maintained in specific-pathogen-free (SPF) facilities at the Institute of Pathology of the University of Bern. All procedures were performed in accordance with ethical guidelines and approved animal license protocols of the Canton of Bern (BE3/18 and BE31/2021). Mice were housed with a 12 h light/dark cycle, regulated temperature and humidity, and provided unlimited access to food and water. C57BL/6J, CD45.1 (B6.SJL-PtprcaPepcb/BoyJ), Mafb-Cre (Mafbtm1.1(cre)Kmm/J), and R26R-EYFP (B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J) mice were purchased from The Jackson Laboratory and bred in-house. IL-1RaLysM mice had been generated (31) by crossing IL-1Rafl/fl (Il1rntm1.1Cga) mice (31) to LysM-Cre (Lyz2tm1(cre)Ifo/J) mice (32) and were kindly provided by Marc Donath (Department of Biomedicine, University Hospital Basel). To obtain IL-1RaMafb and IL-1RaCD11c mice, we crossed IL-1Rafl/fl mice with Mafb- Cre or CD11c-Cre (B6.Cg-Tg(Itgax-cre)1-1Reiz) (34) mice, respectively. CD11c-Cre mice were a gift from Manfred Kopf (Institute of Molecular Health Sciences, ETH Zurich). In general, IL-1Rafl/fl Cre-negative littermates served as controls; for some experiments IL- 1Rawt/wt Cre-positive mice were used as controls and yielded wild type results. R26R- EYFP mice (termed EYFPLSL here) were crossed with LysM-Cre, Mafb-Cre, or CD11c-Cre mice to create the respective EYFP-Cre-reporter strains, which we referred to as EYFPLysM, EYFPMafb, or EYFPCD11c here. All mouse strains were backcrossed onto C57BL/6 for more than 10 generations or had been generated on a C57BL/6 background. Age- and sex-matched animals were randomly assigned to experimental groups. C. albicans (SC5413) was grown in YPD medium (BD DifcoTM YPD Broth, BD Sciences) at 30°C for 18 hours. C. albicans yeast cells were washed twice in sterile PBS and counted using a hemocytometer. To obtain C. albicans hyphae, washed C. albicans yeast were cultured in RPMI 1640 (10% FBS) at 37°C for 4 h. Heat-killed yeast and hyphae were prepared by incubation at 72°C for 1 h, and complete inactivation was confirmed by plating on YPD agar plates at 30°C for 48 h. For in vivo experiments, eight- to ten-week-old mice were infected intravenously with 0.5 - 2.5x105 colony forming units (CFU) C. albicans via the lateral tail vein. Kidneys were disrupted in 0.5 to 1 ml of TRIzol Reagent (Ambion Life Technologies) with stainless steel beads (5 mm; Qiagen) in a TissueLyser II (Qiagen) for 2 cycles of 2 min at 25Hz. Total mRNA was isolated according to manufacturer's instructions. Contaminating DNA was digested by RNase-free DNase (Life Technologies), mRNA concentrations were measured with a NanoDropTM One spectrophotometer (Thermo Fisher) and 1 μg mRNA/reaction was reverse-transcribed using GoScriptTM Reverse Transcriptase (Promega) in presence of RNase inhibitor (Bioconcept). Quantitative PCR was performed using KAPA SYBR® FAST qPCR Master Mix (2X) Kit (Sigma) on a StepOnePlusTM Real-Time PCR System (Thermo Fisher), and expression was normalized to G6pdx or Actb expression. For FACS-sorted cells, RNA was purified using the ReliaPrepTM RNA Miniprep Systems (Promega) following the manufacturers' instructions. Kidneys of infected mice were harvested at day 2 post infection, snap-frozen in liquid nitrogen before homogenization in TRI Reagent® (Zymo Research) in BashingBeatTM lysis tubes (Zymo Research) with a TissueLyser II (Qiagen) for 3 cycles of 1 min at 30Hz. Total RNA was extracted using the Direct-zolTM RNA miniprep Plus kit (Zymo Research) and stored at -80°C until use. The quantity and quality of the purified total RNA was assessed using a Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Thermo Fisher) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Agilent), respectively. Two hundred ng of input RNA was first depleted of ribosomal RNA and globin mRNA using RiboCop for HMR + Globin Depletion Kit (Lexogen) following the manufacturer's instructions. Thereafter, cDNA libraries were generated using a CORALL Total RNA-Seq V2 library Prep.kit with UDIs 12nt set A1 (Lexogen) according to the protocol for long insert sizes. The quantity and length of the cDNA libraries were investigated using the Qubit 4.0 fluorometer and Advanced Analytical Fragment Analyzer System as above. Library quantification was also determined using a JetSeq library Quantification Lo-ROX kit (Bioline) following the manufacturer's instructions.