title: GSM4504085: bTB PCR-N PPD-B rep1; Bos taurus; RNA-Seq
library_construction_protocol: About 50 ml of whole blood with anticoagulant (heparin) was collected aseptically from each animal. PBMCs were isolated using bovine lymphocyte-separation medium (Solarbio, China) by density-gradient centrifugation, per the manufacturer's instructions. Red blood cells (RBC) were removed by RBC lysis, the cells were washed by PBS for three times. And then cells from each animal were counted and seeded into 12-well tissue culture plates at 5×106 cells/ml OpTmizer™ T-Cell Expansion SFM (Gibico) containing 1% L-Glutamine (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich, Ltd.). Subsequently, the PBMCs were stimulated with 300 IU PPD-B (3000 IU/mL, Prionics AG, Schlieren, Switzerland); unstimulated PBMCs (PBS-treated) from the same cattle were run as a negative control. Subsequently, PBMCs were incubated at 37℃ with 5% CO2, harvested for treatment 6 h post-stimulation. Total RNA was extracted using the TRIzol reagent.  TruSeq Stranded mRNA sample prep kit (Illumina, San Diego, USA) was used with 3 ug of total RNA for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols
attributes.tag: GEO Accession
attributes.value: GSM4504085