Gene expression data related to Broche et al.: "Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression"

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Jeltsch, Albert; Broche, Julian, 2023, "Gene expression data related to Broche et al.: "Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression"", https://doi.org/10.18419/darus-2813, DaRUS, V1

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Description	In the publication "Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression" bacterial DNA-adenine N6-Methyltransferases were expressed in human HEK293 cells. Cell proliferation and gene expression were investigated. Gene expression was investigated by RNA-seq. RNA-seq data were obtained from stable HEK293 cell lines, containing the bacterial N6-MTase CcrM (wildtype or catalytically inactive D39A mutant). Expression of CcrM was controlled by the doxycycline (dox)-inducible TRE3G promoter. RNA-seq experiments were performed with cells before ('no dox') or after 10 days ('10d dox') of dox treatment. Total RNA was isolated from ~10^6 cells using the RNeasy® Mini Kit (Qiagen), and quantified via NanoDrop1000. For each library, 1 µg RNA was taken as input material. Since total RNA is mostly constituted of rRNA (≥ 80%), poly-adenylated mRNA was first enriched using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB). Afterwards, RNA-seq libraries were generated using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB), in combination with NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB), thus resulting in strand-specific RNA libraries (opposite sense strand). For quality assessment of the RNA-seq libraries, fragment size distributions and concentrations were determined by Labchip® GX Touch (Perkin Elmer). Finally, 300 ng of each RNA library was submitted to Novogene Corporation Inc. U.K. for Next-Generation-Sequencing (Illumina Novaseq 6000 system, paired-end, 2 x 150bp-mode). Sequencing data were received in FASTQ format (~2 x 20 million reads/sample), and all subsequent data processing steps were performed on the European Galaxy Platform (Afgan et al., 2016).
Subject	Medicine, Health and Life Sciences
Keyword	Gene Expression, DNA Modification, DNA Methylation
Related Publication	Broche et al., Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression, Communications Biology, in press.
License/Data Use Agreement	CC BY 4.0

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Citation Metadata

Persistent Identifier	doi:10.18419/darus-2813
Publication Date	2023-01-10
Title	Gene expression data related to Broche et al.: "Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression"
Author	Jeltsch, Albert (Universität Stuttgart) - ORCID: 0000-0001-6113-9290 Broche, Julian (Universität Stuttgart) - ORCID: 0000-0003-3713-5462
Point of Contact	Use email button above to contact. Jeltsch, Albert (Universität Stuttgart)
Description	In the publication "Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression" bacterial DNA-adenine N6-Methyltransferases were expressed in human HEK293 cells. Cell proliferation and gene expression were investigated. Gene expression was investigated by RNA-seq. RNA-seq data were obtained from stable HEK293 cell lines, containing the bacterial N6-MTase CcrM (wildtype or catalytically inactive D39A mutant). Expression of CcrM was controlled by the doxycycline (dox)-inducible TRE3G promoter. RNA-seq experiments were performed with cells before ('no dox') or after 10 days ('10d dox') of dox treatment. Total RNA was isolated from ~10^6 cells using the RNeasy® Mini Kit (Qiagen), and quantified via NanoDrop1000. For each library, 1 µg RNA was taken as input material. Since total RNA is mostly constituted of rRNA (≥ 80%), poly-adenylated mRNA was first enriched using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB). Afterwards, RNA-seq libraries were generated using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB), in combination with NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB), thus resulting in strand-specific RNA libraries (opposite sense strand). For quality assessment of the RNA-seq libraries, fragment size distributions and concentrations were determined by Labchip® GX Touch (Perkin Elmer). Finally, 300 ng of each RNA library was submitted to Novogene Corporation Inc. U.K. for Next-Generation-Sequencing (Illumina Novaseq 6000 system, paired-end, 2 x 150bp-mode). Sequencing data were received in FASTQ format (~2 x 20 million reads/sample), and all subsequent data processing steps were performed on the European Galaxy Platform (Afgan et al., 2016).
Subject	Medicine, Health and Life Sciences
Keyword	Gene Expression https://www.wikidata.org/wiki/Q26972 (Wikidata) DNA Modification https://www.wikidata.org/wiki/Q14877610 (Wikidata) DNA Methylation https://www.wikidata.org/wiki/Q874745 (Wikidata)
Topic Classification	Biochemistry (LCSH) http://id.loc.gov/authorities/subjects/sh85014171 Molecular Biology (LCSH) https://id.loc.gov/authorities/subjects/sh85086577.html General Genetics and Functional Genome Biology (DFGFO) https://github.com/tibonto/dfgfo/201-05
Related Publication	Broche et al., Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression, Communications Biology, in press.
Production Date	2021-11-02
Production Location	Novogene Co., Ltd. (NGS services)
Contributor	Other : Novogene Co., Ltd. (UK)
Funding Information	University of Stuttgart: Terra Incognita program DFG: JE 252/35-1
Depositor	Jeltsch, Albert
Deposit Date	2022-04-13
Data Type	Next-Generation Sequencing data, RNA-seq
Did it work?	Yes
Explanation	Analysis of the generated RNA-seq data confirmed the high reproducibilty among replicates and an overall satisfying sequencing quality. The obtained data revealed that genome-wide deposition of m6dA leads to the differential expression of around 100 genes, thus providing further evidence for the impact of this modification on gene regulation.

Process Metadata

Processing Methods	Quality control (The quality of the reads was analyzed using the FastQC tool.) Trimming of Illumina adapters (Trimming of Illumina adapters was conducted using the Trimmomatic tool using the options: ILLUMINA-CLIP step; TrueSeq3 paired-end for MiSeq and HiSeq) ; Parameters: Initial ILLUMINACLIP step, Adapter sequences to use Mapping (Paired-end reads were mapped on the human reference genome Hg38 by utilizing the gapped-read mapper RNA STAR in default settings.) Generation of Count tables (The obtained BAM files, as well as the refFLAT transcript file (GTF format), served as input to generate count tables using the HTseq-count tool in ‘reverse strand‘ mode.) ; Parameters: Stranded DIfferential gene expression analysis (Differential gene expression testing was conducted by employing DeSeq2, using the previously obtained HTseq-count tables. DeSeq2 utilizes the median-of-ratios method for normalization. The resulting normalized counts file was taken as a basis for data depiction.) Gene set enrichment analysis (Differentially expressed genes were called on the basis of p<0.05 after Benjamini & Hochberg p-value correction for multiple testing. Gene set enrichment analysis was conducted using the Enrichr Tool.)
Method Parameters	Initial ILLUMINACLIP step : Yes Adapter sequences to use : TrueSeq3 (paired-ended, for MiSeq and HiSeq) Stranded : Reverse
Software	FastQC Version: Galaxy Version 0.73+galaxy0 (https://usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/devteam/fastqc/fastqc/0.73+galaxy0) GPL v3 or later (Andrews, S. (n.d.). FastQC A Quality Control tool for High Throughput Sequence Data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) Trimmomatic Version: Galaxy Version 0.38.1 (https://usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/pjbriggs/trimmomatic/trimmomatic/0.38.1) (Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114–2120. doi: 10.1093/bioinformatics/btu170) RNA STAR Version: Galaxy Version 2.7.8a+galaxy1 (https://usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/iuc/rgrnastar/rna_star/2.7.8a+galaxy1) MIT (Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., & Gingeras, T. R. (2012). STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29(1), 15–21. doi: 10.1093/bioinformatics/bts635) HTseq-count Version: Galaxy Version 0.9.1+galaxy1 (https://usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/lparsons/htseq_count/htseq_count/0.9.1+galaxy1) GPL 3.0 (Anders, S., Pyl, P. T., & Huber, W. (2014). HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics, 31(2), 166–169. doi: 10.1093/bioinformatics/btu638) DESeq2 Version: Galaxy Version 2.11.40.7+galaxy2 (https://usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.7+galaxy2) LGPL doi: 10.18129/B9.bioc.DESeq2 (Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15(12). doi: 10.1186/s13059-014-0550-8) Enrichr Tool (https://maayanlab.cloud/Enrichr/) (Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, Koplev S, Jenkins SL, Jagodnik KM, Lachmann A, McDermott MG, Monteiro CD, Gundersen GW, Ma'ayan A. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Research. 2016; doi: 10.1093/nar/gkw377.)
Instruments	Novaseq 6000 (Sequencing platform) , Location: Novogene Co., Ltd. (UK)
Processing Steps	1. Analysis ; Methods: Quality of reads ; Software: FastQC 2. Postprocessing ; Methods: Trimming of Illumina adapters ; Software: Trimmomatic 3. Postprocessing ; Methods: Mapping on human reference genome Hg38 ; Software: RNA STAR ; Output: BAM files, refFLAT transcript file (GTF format) 4. Analysis ; Methods: Generate count tables ; Software: HTseq-count ; Input: BAM files, refFLAT transcript file (GTF format) ; Output: HTseq-count tables 5. Analysis ; Methods: Differential gene expression testing ; Error Method: p < 0.05, Benjamini & Hochberg p-value correction for multiple testing ; Software: DeSeq2 ; Input: HTseq-count tables ; Output: normalized counts file 6. Analysis ; Methods: Gene set enrichment ; Software: Enrichr Tool

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