2024-08-29
ENA first public
ERC000011
ENA-CHECKLIST
UCD School of Agriculture and Food Science, University College Dublin, Belfield, D04 V1W8, Dublin, Ireland
INSDC center name
public
INSDC status
ERS20882626
SRA accession
E-MTAB-14366:R4S2
Submitter Id
ArrayExpress
broker name
WC1.2+ γδ T
cell_type
not collected
collection date
cattle
common name
Protocols: 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.
description
Tuberculosis, Bovine
disease
not collected
geographic location (country and/or sea)
Cattle 4
individual
not applicable
isolate
Bos taurus
organism
Bos taurus
organism
blood
organism part
TB Infected
phenotype
Bos taurus
scientific_name
PPDB
stimulus
Holstein-Friesian
strain
R4S2
title