strain: wild-type H37Rv Stage: anaerobiosis at NRP-2 stage in the dormancy model
Treatment protocol
Bacteria were incubated on ice for 1 min and then centrifuged at 12,000 g for 30 s. The cell pellets were added with fresh chloroform/methanol (3:1, v/v) at a rate of 200 μl per OD, votexed for 20 s, and immediately mixed well with 5 vol Trizol (Takara, Dalian, China).
Growth protocol
Wild-type H37Rv and Δdgc strains were grown at 37oC in 7H9-OADC-Tween 80 medium under aerobic conditions or in an in vitro dormancy model with methylene blue by rapid shaking. Bacteria were collected at OD600 =1.3 for the aerobic cultures and upon the beginning of complete decolorization of methylene blue (a oxygen sensitive indicator) for the cultures in the dormancy model.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low RNA Input Linear Amplification kit (Cat#5184-3523, Agilent technologies, Santa Clara, CA, US), 5-(3-aminoallyl)-UTP (Cat#AM8436, Ambion, Austin, TX,US), Cy3 NHS ester (Cat#PA13105,GE healthcare Biosciences, Pittsburgh, PA,US) followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), Stabilization and Drying Solution (Cat#5185-5979, Agilent technologies, Santa Clara, CA, US) followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565BA, Agilent technologies, Santa Clara, CA, US) and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) with default settings，Scan resolution=5μm, PMT 100%，10%.
Description
Gene expression upon the beginning of anaerobiosis at NRP-2 stage in the dormancy model
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
