GSE135818
RNA-seq of Aspergillus fumigatus infected to HL-60 derived macrophage-like cells for 0,1, and 2 hours.
Purpose : The goals of this study are to compare transcriptome profiling (RNA-seq) of Aspergillus fumigatus infected to macrophage-like cells for 0, 1 and 2 hours. Method : HL-60 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% Gibco™ Antibiotic-Antimycotic at 37°C with 5% CO2. To induce the differentiation of HL-60 cells into macrophage-like cells, HL-60 cells were stimulated with 50 nM 12-o-tetradecanoylphorbol-13-acetate (PMA) for 2 days. A total of 10^3 A.fumigatus conidia were transferred to 5 × 10^4 differentiated HL-60 cells in RPMI 1640 medium supplemented with 5% FBS. After incubating for 0, 1, and 2 hrs, the sample tubes were vigorously agitated with TRIzol® (Invitrogen) containing glass beads to extract total RNA of alive A.fumigatus. ). Libraries were prepared from 2 µg of total RNA using a SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc., USA). Isolation of mRNA was performed using a Poly(A) RNA Selection Kit (LEXOGEN, Inc., Austria). The isolated mRNAs were used for cDNA synthesis and shearing, following the manufacturer's instructions. Indexing was performed using the Illumina indexes 1-12. An enrichment step was carried out using PCR. Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit with a StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 bp sequencing using a HiSeq 2500 (Illumina, Inc., USA). mRNA-seq reads were mapped using the TopHat software (Cole Trapnell et al., 2009) tool to obtain an alignment file. Overall design: A.fumigatus conidia were infected into macrophage-like cells for 0,1 and 2hours and total RNA of A.fumigatus was extracted respectively. Their transcriptome expression patterns were compared by RNA-seq.