biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsVasileios BekiarisNextSeq 2000RNA-seq of coding RNAMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16689This experiment was designed to explore the biological relevance of the transcription factor Tbet in intestinal γδT17 cells. For this purpose, we crossed TbetF/F with RORγtCRE-R26LSLRFP mice, where RFP expression in the ROSA26 locus was induced by RORγt-driven Cre recombinase. We then sorted Tbet-deficient γδT17 cells (RORγtRFP+) from lamina propria and performed bulk RNA-seq.biostudies-arrayexpressLibrary Construction - The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from approximately 1 ng total-RNA. Double stranded cDNA was amplified by LD PCR (11 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). Thereby 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step 1:5 dilutions of the unique dual indexing (i7 and I5) adapters were used. The libraries were quantified using the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA).Sample Collection - Lymphocytes were isolated from small intestine and colon lamina propria of RORγtCRE-TbetF/F mice. 4 mice were pooled in each biological repeat. Subsequently cells were stained and sorted as CD8−CD4−CD19−TCRβ−CD45+CD3+TCRγδ+RFP+ using a FACSAriaIII sorter and collected directly in RNAprotect Cell Reagent (Qiagen).Nucleic Acid Extraction - Total RNA was extracted from sorted cells stabilized in RNAprotect buffer according to the “Purification of total RNA from animal and human cells” protocol of the RNeasy Plus Micro Kit (QIAGEN, Hilden, Germany). In brief, cells were stored and shipped in buffer RNAprotect at 2-8 °C. After pelleting by centrifugation for 5 minutes at 5,000 x g, the RNAprotect was replaced by 350 µl buffer RLT Plus and the samples were homogenized by vortexing for 30 seconds. Genomic DNA contamination was removed by using gDNA Eliminator spin columns. Next one volume of 70 % ethanol was added and the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Finally the total RNA was eluted in 12 μl of nuclease free water. Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico LabChip reagent set (Agilent, Palo Alto, CA, USA).Sequencing - Equimolar amounts of each library were sequenced on an Illumina NextSeq 2000 instrument controlled by the NextSeq 2000 Control Software (NCS) v1.4.1.39716, using one 50 cycles P3 Flow Cell with the dual index, single-read (SR) run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA) v3.9.25. The resulting .cbcl files were converted into .fastq files with the bcl2fastq v2.20 software.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesVasileios BekiarisfalseBulk RNA-seq of murine γδT17 cells isolated from the lamina propria of Tbet-deficient miceThis experiment was designed to explore the biological relevance of the transcription factor Tbet in intestinal γδT17 cells. For this purpose, we crossed TbetF/F with RORγtCRE-R26LSLRFP mice, where RFP expression in the ROSA26 locus was induced by RORγt-driven Cre recombinase. We then sorted Tbet-deficient γδT17 cells (RORγtRFP+) from lamina propria and performed bulk RNA-seq.2026-03-14T00:00:00Z2026-03-14T02:03:47.002Z2026-02-26T14:10:31.044ZE-MTAB-16689ERP189676E-MTAB-16691E-MTAB-16692E-MTAB-16690EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184