Bulk RNA-seq of murine γδT17 and γδT1 cells from the lamina propria and peripheral lymph nodes of wild-type mice
Bulk RNA-seq of murine γδT17 and γδT1 cells from the lamina propria and peripheral lymph nodes of wild-type mice
Lymphocytes were isolated from small intestine, colon lamina propria and peripheral lymph nodes of wild type mice that transgenically expressed GFP and AmCyan under the control of the RORγt or Tbet promoters respectively (RORγtGFPTbetAMCYAN), 4 mice were pooled in each biological repeat, n=3. Subsequently cells were sorted as GFP+ (RORγt+), AmCyan+ (Tbet+) or GFP+AmCyan+ (RORγt+Tbet+) using a FACSAriaIII sorter and collected directly in RNAprotect Cell Reagent (Qiagen).  Total RNA was extracted from sorted cells 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 RNAlater at 2-8 °C. After pelleting by centrifugation for 5 minutes at 5,000 x g, the RNAlater was replaced by 350 µl buffer RLT Plus and the samples were homogenized by vortexing for 30 sec. Genomic DNA contamination was removed 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 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 500 pg total-RNA. Double stranded cDNA was amplified by LD PCR (12 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). 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 index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). 