title: RNA-seq of murine Vg4+CD27-CD44+ and Vg4-CD27-CD44+ gamma delta from the lymph nodes of retinoic acid receptor deficient mice and littermate controls
design: RNA-seq of murine Vg4+CD27-CD44+ and Vg4-CD27-CD44+ gamma delta from the lymph nodes of retinoic acid receptor deficient mice and littermate controls
library_construction_protocol: Lymphocytes were isolated from peripheral, cervical and auricular LNs of RORγtCRE-RARdnF/F or littermate control mice and γδ T cells were enriched as described above. Subsequently cells were stained as described above and Vγ4+TCRγδ+CD3+CD44+CD27− and Vγ4−TCRγδ+CD3+CD44+CD27− populations from each mouse strain were sorted using a FACSAriaIII: populations into RNA protect (QIAGEN, Hilden, Germany).  Total RNA was extracted from the sorted cells according to the “Purification of total RNA from animal and human cells” protocol of the RNeasy Plus Micro Kit (QIAGEN). After pelleting by centrifugation for 5 minutes at 5,000 x g, the RNA protect 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 100 pg total-RNA. Double stranded cDNA was amplified by LD PCR (13 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).