title: Comparative analysis of WC1.1+ and WC1.2+ γδ T cell subset responses from cattle naturally infected with Mycobacterium bovis to repeat stimulation with mycobacterial antigens
design: Comparative analysis of WC1.1+ and WC1.2+ γδ T cell subset responses from cattle naturally infected with Mycobacterium bovis to repeat stimulation with mycobacterial antigens
library_name: R4S2_p
library_construction_protocol: Male Holstein-Friesian (B. taurus) cattle were used for this study. Animals (n=5) were selected from a herd of animals naturally infected with M. bovis maintained at the Department of Agriculture, Food and Marine research farm in Longtown, Co. Kildare, Ireland. The infected animals had tested positive for bovine tuberculosis by single intradermal comparative tuberculin test (SICTT) and also by the whole blood interferon (IFN)-γ release assay (IGRA, University College Dublin, Ireland). Peripheral blood from the jugular vein of bTB infected animals was collected in 9 ml vacutainer tubes containing sodium heparin anticoagulant. The whole blood was stimulated with bovine purified protein derivative (PPDb) (10 μg/ml) for 6 hours in a humidified atmosphere with 5% CO2 at 37° C. Non-stimulated whole blood cultures were also included as controls. At the end of incubation, PBMCs from each culture were harvested by density gradient centrifugation using Histopaque (Density 1.077 g/mL; Sigma-Aldrich). PBMCs were washed twice in PBS and then subjected to magnetic cell separation of γδ T cells, WC1.1+ and WC1.2+ subsets using primary monoclonal antibodies (mAbs) from Washington State University Monoclonal Antibody Center (WSUMAC), Pullman, WA, USA and anti-mouse IgG magnetic microbeads (Miltenyi Biotec, UK) according to manufacturer protocols. The primary mAbs used in this study were GB21A, an IgG2b isotype with catalogue number BOV2058, which is specific to the γδ TCR. For the WC1.1+ marker, the mAb BAQ89A, an IgG1 isotype with catalogue number BOV2055, was utilised. The mAb CACTB32A, an IgG1 isotype with catalogue number BOV2054, was also used to target the WC1.2+ marker. Isolated cell samples were washed in PBS, pelleted in microfuge tubes, and stored at -80°C. Total RNA was extracted from the cell pellets using AllPrep DNA/RNA Mini Kit (Qiagen) per the manufacturer's instructions. RNA quantity, integrity, and purity were assessed using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies Ltd., Cork, Ireland), according to the manufacturer's instructions. All RNA samples used for transcriptomic analysis (n = 5) had RNA integrity number (RIN) values >7.5.  After determining RNA integrity and quality, RNA was prepared and sequenced at Eurofins Genomics (Constance, Germany) using an INVIEW Transcriptome Discover product. This included purification of mRNA, fragmentation, strand-specific cDNA synthesis, end-repair, ligation of sequencing adapters, amplification and purification.
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-14366:R4S2_p