The murine macrophage cell line J774A.1 (American Type Culture Collection, ATCC) was used in this study. J774 cells were cultured in DMEM (HyClone from Thermo Scientific) medium containing 10% (v/v) fetal calf serum, 50 μg/ml of penicillin/streptomycin and 2 mM glutamine. Cells were used to conduct experiments when they reached ~70% confluence. All treatments were performed in serum-free medium. All mycobacteria were grown on Middlebrook 7H11 agar at 37°C, 5% CO2-95% air atmosphere. For broth cultures, H37Ra (ATCC 25177, the lab strain of MTB) and BCG (ATCC 35734, the vaccine strain of M. Bovis) were grown in 7H9 medium supplemented with glycerol (0.5%, vol/vol) and OADC supplement. M. smegmatis (mc2-155, ATCC 700084) was grown in 7H9 medium supplemented with glycerol (0.5%, vol/vol) and ADS supplement. All liquid cultures were supplemented with 0.05% Tween 80.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol, followed by on-column digestion of DNA using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA quantity and quality were assessed with a Qubit RNA Assay Kit using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).
Label
biotin
Label protocol
Standard Affymetrix 3' IVT Express Kit
Hybridization protocol
Standard Affymetrix 3' IVT Express Kit
Scan protocol
Affymetrix 7G scanner
Description
Gene expression data from murine macrophage after infection
Data processing
.chp files were generated by Expression Console (Version 1.1, Affymetrix, Santa Clara, CA, USA) using global normalization to a target intensity of 200 by the MAS5 algorithm
