Bulk RNA Sequencing of Regulatory T Cells

FACS-sorted tTreg from WT-Foxp3^RFP and Kcnk18^G339R/Foxp3^RFP thymus were used for bulk RNA sequencing. RNA was isolated with Quick-RNA Microprep Kit (Zymo Research) as described above. Quality and amount of RNA were verified by NanoDrop and Bioanalyzer RNA 6000 Nano Kit (Agilent). Samples with RIN values > 6.5 were used for RNA sequencing. NEBNext^® rRNA depletion was performed followed by NEBNext directional Ultra RNA II Library preparation and sequencing on NextSeq500 (Illumina) platform (75 cycles, high output v2 kit). Raw sequencing data were analyzed by Linux bash tools following the analysis pipeline: (1) quality control (fastqc), (2) trimming (Trimmomatic 0.36),^{61 (link)} (3) alignment to mouse genome (Hisat 2.1.0; build: mm10),^{62 (link)} (4) aligned read sorting (Samtools)^{63 (link)} and (5) read counting (HTseq 0.10.0.^{64 (link)}) Expression analysis was performed with R/Bioconducter DESeq2.^{65 (link)} Treg signature genes were assigned according to a previous study.^{66 (link)} Significantly regulated genes (FDR < 0.05) were used for further analysis and visualization using pHeatmap (https://cran.r-project.org/package=pheatmap) package in R. GO term gene enrichment analysis was performed using the PANTHER classification system online tool.^{67 (link),68 (link)}

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Ruck T., Bock S., Pfeuffer S., Schroeter C.B., Cengiz D., Marciniak P., Lindner M., Herrmann A., Liebmann M., Kovac S., Gola L., Rolfes L., Pawlitzki M., Opel N., Hahn T., Dannlowski U., Pap T., Luessi F., Schreiber J.A., Wünsch B., Kuhlmann T., Seebohm G., Tackenberg B., Seja P., Döring F., Wischmeyer E., Chasan A.I., Roth J., Klotz L., Meyer zu Hörste G., Wiendl H., Marschall T., Floess S., Huehn J., Budde T., Bopp T., Bittner S, & Meuth S.G. (2021). K2P18.1 translates T cell receptor signals into thymic regulatory T cell development. Cell Research, 32(1), 72-88.

Publication 2021

Quick-RNA Microprep Kit NanoDrop Bioanalyzer RNA 6000 Nano Kit NextSeq500

Bulk Gene Genome Library Mouse Rna ii Rrna Thymus

Corresponding Organization : Heinrich Heine University Düsseldorf

Other organizations : University of Münster, University Medical Center of the Johannes Gutenberg University Mainz, Johannes Gutenberg University Mainz, Philipps University of Marburg, University of Helsinki, University of Würzburg, Helmholtz Centre for Infection Research, Focus (Germany)

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Variable analysis

independent variables

Genotype (WT-Foxp3^RFP and Kcnk18^G339R/Foxp3^RFP)

dependent variables

Gene expression profiles of tTreg cells from WT-Foxp3^RFP and Kcnk18^G339R/Foxp3^RFP thymus

control variables

Samples with RIN values > 6.5 were used for RNA sequencing

controls

No positive or negative controls are explicitly mentioned in the protocol.

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