Bulk RNA-seq of murine γδT17 cells isolated from the lamina propria of wild-type mice at day 0 and day 7
Bulk RNA-seq of murine γδT17 cells isolated from the lamina propria of wild-type mice at day 0 and day 7
Lymphocytes were isolated from small intestine and colon lamina propria of wild type RORγtGFP reporter mice at day 0 and day 7.  4-8 mice were pooled in each biological repeat. Subsequently cells were stained and sorted (pooling siLP and cLP) as CD8−CD4−TCRβ−CD45+CD3+TCRγδ+GFP+ using a FACSAriaIII sorter, and collected directly in RNAprotect Cell Reagent (Qiagen). Note: Some mice also carried Amcyan and/or RFP reporter alleles. These channels were not used for gating or population selection.  Total RNA was extracted from FACS sorted mouse 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).  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). 