GSE170750
Next Generation Sequencing  of Wild Type pDC Transcriptomes in a longitudinal activation study (CpG 0h, 2h, 6h, 12)
Purpose: The goals of this study are to compare NGS-derived pDC transcriptome profiling (RNA-seq) normalized counts and differential expression of genes between different pDC states (steady state, or TLR9 activated for 2h, 6h, or 12h with CpG) in Batf presence and absence. Methods: mRNA profiles of Bone marrow-derived Flt3-L cultured FACS purified pDCs from wild-type and Batf-knockout mice that were left naive or stimulated with TLR9 agonist CpG for 2h, 6h r 12h were generated by deep sequencing, in triplicate, using the Illumina HiSeq3000 platform. DNase digested total RNA samples used for transcriptome analyses were quantified (Qubit RNA HS Assay, Thermo Fisher Scientific) and quality measured by capillary electrophoresis using the Fragment Analyzer and the 'Total RNA Standard Sensitivity Assay' (Agilent Technologies, Inc. Santa Clara, USA). All samples in this study showed high quality RNA Quality Numbers (RQN; mean = 9.9). The library preparation was performed according to the manufacturer's protocol using the Illumina® 'TruSeq Stranded mRNA Library Prep Kit'. Briefly, 200 ng total RNA were used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation and library amplification. Bead purified libraries were normalized and finally sequenced on the HiSeq 3000/4000 system (Illumina Inc. San Diego, USA) with a read setup of SR 1x150 bp. The bcl2fastq tool was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing. Results: Data analyses on fastq files were conducted with CLC Genomics Workbench (version 11.0.1, QIAGEN, Venlo. NL). The reads of all probes were adapter trimmed (Illumina TruSeq) and quality trimmed (using the default parameters: bases below Q13 were trimmed from the end of the reads, ambiguous nucleotides maximal 2). Mapping was done against the Mus musculus (mm10; GRCm38.86) (March 24, 2017) genome sequence. After grouping of samples (three biological replicates each) according to their respective experimental condition, multi-group comparisons were made and statistically determined using edgeR on usegalaxy.org The Resulting ?? values were corrected for multiple testing by FDR. A ?? value of =0.05 was considered significant. Conclusions: Our study represents the first detailed analysis of Batf-dependent gene expression in naive and activated pDC transcriptomes in a longitduinal study. Our results show that Batf absence significantly altered the global gene expression patterns in pDCs, modulating many biological pathways important for cell development and effector function. Overall design: mRNA profiles of Bone marrow-derived Flt3-L FACS purified pDCs from wild type and Batf-deficient mice that were left naïve or stimulated with TLR9 agonist CpG for 2h, 6h or 12h.