Illumina HiSeq 4000 paired end sequencing; The protective effect of inflammatory monocytes during systemic C. albicans infection is dependent on collaboration between C-type lectin-like receptors
The protective effect of inflammatory monocytes during systemic C. albicans infection is dependent on collaboration between C-type lectin-like receptors
BIOgel recruited inflammatory cells were recovered by peritoneal lavage and washed as described above. Cells were stained with antibodies against Ly6G, CD4, CD8, Ly6C, F4/80, CD19, DX5 and CD11b. Ly6ChiLy6G-CD11b+F4/80+CD4-CD8-DX5-CD19- inflammatory monocytes were purified by cell sorting on a FACS Aria cell sorter.  Candida albicans SC5314 (ATCC) was plated on YPD agar, cultured for 16-20 h in YPD broth, washed three times with PBS and resuspended at the required concentration in PBS.  Inflammatory monocytes were incubated at 37 degreesC with C. albicans at 3:1 Cells:Candida ratio for 3 h in RPMI containing 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 0.5% HEPES and 50uM 2-mercaptoethanol. Cell pellets were snap frozen and stored in -80 degreesCC.   RNA was extracted using a RNeasy mini kit (Qiagen). Total RNA quality and quantity was assessed using Agilent 2100 Bioanalyser and a RNA Nano 6000 kit  (Agilent Technologies). Total RNA with a RIN value >7 was depleted of ribosomal RNA using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat), (New England BioLabs, NEB) and the sequencing libraries were prepared using the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB).
Experimental Factor: genotype
Dectin-1; Dectin-2 and Mincle triple knockout
Experimental Factor: infect
Candida albicans SC5314