Tiling array

The genomes of haploid wild-type, cell-fusion, and mutant strains were analyzed using a tiling array (Agilent Technologies, Santa Clara, CA, USA). Genomic DNA was extracted using spin columns (Genomic DNA Buffer Set No. 19060, and Genomic tip 500/G No. 10262; Qiagen, Hilden, Germany). A. thaliana genomic DNA was extracted using spin columns (DNeasy Plant Mini Kit No. 69104; Qiagen). For tiling-array analysis, array comparative genomic hybridization (aCGH) was performed using a S. cerevisiae or A. thaliana custom microarray (designed by eArray; Agilent Technologies) for genome-wide copy number analysis. Genomic DNA (100 ng) was fluorescently labeled using the SureTag DNA Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Prior to hybridization, sample and control probes were incubated with the blocking agent and hybridization buffer contained in the Oligo aCGH/ChIP-on-chip Hybridization Kit (Agilent Technologies) at 95 °C for 3 min followed by 37 °C for 3 min. Hybridization was performed at 65 °C for 24 h in a microarray hybridization oven (Agilent Technologies) according to the manufacturer instructions, and the slides were washed with the Oligo aCGH/ChIP Wash Buffer Kit (Agilent Technologies). Array data were quantified and normalized using Feature Extraction Software (v.11.0.1.1; Agilent Technologies).

Whole genome gene expression analyses were performed. Microarray experiments were performed using a custom Agilent tiling array with 180,000 60-mer oligos evenly tiled across the M. tuberculosis H37Rv genome. Features were extracted from the array images using Agilent Feature Extraction Software (v10.7) with local background correction. Probes were first filtered to only include those covering annotated genes. Intensity values were normalised and analysed using GeneSpring software (version 12.6 GX). Firstly, quantile normalisation was applied across the combined slow and fast growth rate datasets, followed by baseline transformation to the median of all samples and averaging of the expression level of all probes across each open reading frame. A 2-way ANOVA (using a p-value cut-off of P ≤0.05 and growth rate and MGT as conditions) was used to identify significantly differentially expressed genes. A further filter was applied to select genes with at least a two-fold change in gene expression between time-points of interest. Gene lists derived from all pairwise comparisons can be found in the Additional file 1. Raw data are deposited at ArrayExpress [15 (link)] under the accession number E-MTAB-4093.