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Next Generation Sequencing of Wild Type pDC Transcriptomes in a longitudinal activation study (CpG 0h, 2h, 6h, 12)

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE170750 | GEO | 2021/04/01

REPOSITORIES: GEO

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Project description: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-knock out 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 pvalues were corrected for multiple testing by FDR. A pvalue 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.

Project description:Purpose: The goals of this study are to compare the pDC chromatin structure (ATAC-seq) at steady state and at 2h after CpG activation. Methods: Cells were harvested and frozen in culture media containing FBS and 5% DMSO. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37°C water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al., 2013), with some modifications based on (Corces et al., 2017). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems), and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina). Results: Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed, only reads mapping as matched pairs and only uniquely mapped reads (mapping quality >= 1) were used for further analysis. Alignments were extended in silico at their 3’-ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peaks were identified using the MACS 2.1.0 algorithm at a cut off of p-value 1e-7, without control file, and with the –nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq peaks were removed. Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations. For differential analysis, reads were counted in all merged peak regions (using Subread), and the replicates for each condition were compared using DESeq2. Conclusions: Our study represents a detailed analysis of naive and activated pDC chromatin that analyzes the role of the transcription factor BATF for chromatin structure in pDCs.

Project description:We generated C57BL/6 mice lacking Bmp10 and/or Bmp9 utilizing the Cre-loxP system. Briefly, Bmp9 constitutive deletion resulted from the replacement of exon 2 by a neomycin resistance cassette. Because Bmp10 deletion leads to early embryonic lethality, we used the tamoxifen-inducible Cre system to generate Bmp10-cKO mice (Rosa26-CreERT2;Bmp10lox/lox) by crossing Rosa26-CreERT2 mice with Bmp10lox/lox mice that possess loxP sites flanking exon 2. To generate double-KO (DKO) mice, we crossed these Rosa26-CreERT2;Bmp10lox/lox mice with Bmp9-KO mice. At the age of 8 weeks, mice were treated with tamoxifen (Sigma) by intraperitoneal injection once a day for 5 days at a dosage of 50 mg/kg. At the age of 5 months, Wild Type and DKO mouse lung tissue was flash frozen in liquid nitrogen and stored at -80°C. RNA extraction, RNA sample quality assessment, RNA library preparation, sequencing and raw data analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). Total RNA was extracted from frozen tissue using the Qiagen RNeasy Plus Mini kit. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument (or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. Gene counts were calculated from uniquely mapped reads using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The gene hit counts table was then used for downstream differential expression analysis. A differential gene expression analysis between WT and DKO groups of samples was performed using the R-package DESeq2 (Wald test).

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Next Generation Sequencing of Wild Type pDC Transcriptomes in a longitudinal activation study (CpG 0h, 2h, 6h, 12)

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