Supplementary Material

Supplementary TablesSupplementary Table 1. Sequencing statistics for ChIP-seq samples.Supplementary Table 2. Pearson correlation coefficients for ChIP-seq biological replicates based on read counts.Supplementary Table 3. Sequencing statistics for RNA-seq samples and Breakdown of RNA-seq mapping.Supplementary Table 4. Excel file with ChIP-seq data.Supplementary Table 5. Excel file with RNA-seq data.Supplementary Table 6. Excel file with data related to antisense and intergenic transcription.Supplementary Table 7. Excel file with transcriptional unit identification.Supplementary Table 8. Excel file with comparison of RNA-seq data generated in this work with those published in Arnvig et al, 2011.

Supplementary FiguresSupplementary Figure 1. Scatter plots and Pearson correlation coefficients between RNAP and NusA

feature counts in exponential and stationary phase datasets. Supplementary Figure 2. qPCR validation of ChIP-seq data. Supplementary Figure 3. Distribution of exponential and stationary phase RPM values across all M.

tuberculosis functional categories. Supplementary Figure 4. qRT-PCR validation of RNA-seq data. Supplementary Figure 5. Anti-sense transcription in M. tuberculosis in Exp phase. Supplementary Figure 6. Anti-sense transcription in M. tuberculosis in Stat phase. Supplementary Figure 7. Intergenic transcription in M. tuberculosis.

Supplementary Figure 8. Scatter plots indicating the correlation between RNA-seq data generated in this work and those published in Arnvig et al., 2011.

Supplementary Figure 9. Plot of the cumulative RPM for TUs on the forward and reverse strand against TU Start.

SampleTotal

numberof reads

Number of mapped reads

Percentage of totalmapped to M.

tuberculosis H37Rv

RNAP Exp1 4926726 3939436 79.96%

RNAP Exp2 30696990 30425765 99.12%

RNAP Stat1 31345981 29182618 93.10%

RNAP Stat2 37057267 35851534 96.75%

NusA Exp1 35404259 34084824 96.27%

NusA Exp2 59313547 55221532 93.10%

NusA Stat1 38812104 30089464 77.53%

NusA Stat2 67602724 58737351 86.89%

Input Exp 36363550 36252682 99.70%

Input Stat 35408441 35291945 99.67%

Supplementary Table 1. Sequencing statistics for ChIP-seq samples.

Supplementary Table 2. Pearson correlation coefficients for ChIP-seq biological replicates based on read counts.

Samples Pearson correlation coefficient(r2) p-value < 2.2e-16

RNAP Exp1-Exp2 0.61

RNAP Stat1-RNAP Stat2 0.47

NusA Exp1-NusA Exp2 0.97

NusA Stat1-NusA Stat2 0.96

Input Exp-Input Stat 0.98

SampleTotal

numberof reads

Number of mapped

reads

Percentage of totalmapped to M.

tuberculosis H37Rv

Exp1 35991584 33548393 93.21%

Exp2 186213063 162482036 87.26%

Stat1 176077447 156586011 88.93%

Supplementary Table 3

Sequencing statistics for RNA-seq samples.

Sample

% Reads mapped to

forward strand

% Reads mapped to

reverse strand

% Reads mapped to the

ribosomal RNA operon

% Remaining

readsin CDS

% Remainingreads in intergenic regions

Exp1 2.23 2.57 95.20 68.20 31.80

Exp2 2.73 2.72 94.56 55.82 44.18

Stat1 1.19 0.62 98.19 48.80 51.20

Breakdown of RNA-seq mapping.

A)

B)

r2 = 0.67 r2 = 0.59

A B

Supplementary Figure 1. Scatter plots and Pearson correlation coefficients between RNAP and NusA feature counts for all CDS and inter-CDS regions in exponential (A) and stationary phase (B) datasets.

A

B

C

Supplementary Figure 2 (legend on next page).

Supplementary Figure 2. qPCR validation of ChIP-seq data. (A) qPCR validation of RNAP ChIP-seq data obtained in Exp phase. Eight features, including promoter regions (P), IG regions and CDSs were selected for qPCR validation based on their enrichment ratios (ER) in ChIP-seq experiments with RNAP in Exp phase. The table reports full comparison of the log2 values obtained in qPCR after normalization to the input sample with log2 figures calculated from the ChIP-seq profile. The correlation coefficient is indicated on the graph. (B) qPCR validation of RNAP ChIP-seq data obtained in Stat phase. Eight features, including promoter regions (P), IG regions and CDSs were selected for qPCR validation based on their enrichment ratios (ER) in ChIP-seq experiments with RNAP in Stat phase. The table reports full comparison of the log2 values obtained in qPCR after normalization to the input sample with log2 figures calculated from the ChIP-seq profile. The correlation coefficient is indicated on the graph. (C) qPCR validation of NusA ChIP-seq data obtained in Exp phase. Twelve features, including promoter regions (P), IG regions and CDSs were selected for qPCR validation based on their enrichment ratios (ER) in ChIP-seq experiments with NusA in Exp phase. The table reports full comparison of the log2 values obtained in qPCR after normalization to the input sample with log2 figures calculated from the ChIP-seq profile. The correlation coefficient is indicated on the graph.

Supplementary Figure 3. Distribution of exponential and stationary phase RPM values across all M. tuberculosis functional categories. (A) Dynamic range of RPM values in Exp phase. (B) Dynamic range of RPM values in Stat phase.

A B

-4 -2 0 2 4 6 8

-6

-4

-2

0

2

4

6

8

R² = 0.917518979206504

log2 (EXP/STAT) qRT-PCR

log2

(EXP

/STA

T) R

PM

Feature Log2 (EXP/STAT) RPM

Log2 (EXP/STAT) qRT-PCR

rv0250 0.86 3.37

rv0251 -2.99 -2.13

IG rv0250-rv0251

0.48 0.24

5’-UTR rv3648c 3.52 5.93

rv3648c 3.61 4.48

rv0842 -0.29 1.52

rv3806 5.78 5.94

rv2244 5.34 5.89

rv0188 -4.82 -3.39

rv1285 -2.25 -1.16

sigA 2.39 3.33

rrs -0.002 -0.60

Supplementary Figure 4. qRT-PCR validation of RNA-seq data. Twelve features, including CDSs, IG regions and anti-sense transcripts, were selected for validation by qRT-PCR using RNA from Exp and Stat phase. Data were normalized to the total amount of RNA. Results are expressed as log2 of the Exp/Stat ratio calculated from RPM values and from qRT-PCR figures. The correlation coefficient is indicated on the graph.

RNAP Exp

NusA Exp

RNASeq Exp fwd strand

ino1

A B

RNASeq Exp rev strand

RNAP Stat

NusA Stat

RNASeq Stat fwd strand

RNASeq Stat rev strand

C

rv0842

Supplementary Figure 5. Anti-sense transcription in M. tuberculosis in Exp phase. Examples of anti-sense transcription profiles visualized using the UCSC genome browser. The genome annotation is provided between the Exp and Stat tracks. The scale is shown at the top. The scale for the number of reads is displayed on the y-axis. (A) Anti-sense RNA starting in the middle of the ino1 CDS. (B) Anti-sense RNA starting inside rv0842 and extending upstream of the CDS. (C) Anti-sense transcription of rv0061.

rv0061

5,000

5,000

500

500

5,000

5,000

500

500

5,000

5,000

100

100

5,000

5,000

100

100

3,000

1,000

100

100

3,000

1,000

100

100

rv1734c

B

rv3684

A

Supplementary Figure 6. Anti-sense transcription in M. tuberculosis in Stat phase. Examples of anti-sense transcription profiles visualized using the UCSC genome browser. The genome annotation is provided between the Exp and Stat tracks. The scale is shown at the top. The scale for the number of reads is displayed on the y-axis. (A) Anti-sense transcription starting in rv3684. (B) Anti-sense RNA starting downstream of the rv1734c CDS.

RNAP Exp

NusA Exp

RNASeq Exp fwd strand

RNASeq Exp rev strand

RNAP Stat

NusA Stat

RNASeq Stat rev strand

RNASeq Stat fwd strand

3,000

5,000

100

100

3,000

5,000

100

100

20,000

1,000

500

500

4,000

3,000

500

500

CBA

IG rv3218 – rv3219 IG rv3648c – rv3649 IG rv1144 – rv1145

Supplementary Figure 7. Intergenic transcription in M. tuberculosis. Examples of intergenic transcription profiles visualized using the UCSC genome browser. The scale is shown at the top. The scale for the number of reads is displayed on the y-axis. (A) IG between rv3218 and rv3219. The long 5’-UTR of rv3219 is evident. (B) IG between rv3648c and rv3649. The long 5’-UTR of rv3648c corresponds to the RNAP peak. (C) IG between rv1144 and rv1145, where an sRNA is transcribed.

RNAP Exp

NusA Exp

RNASeq Exp fwd strand

RNASeq Exp rev strand

RNAP Stat

NusA Stat

RNASeq Stat fwd strand

RNASeq Stat rev strand

5,000

1,000

100

100

5,000

1,000

100

100

1,000

1,000

100

100

1,000

1,000

100

100

3,000

3,000

100

100

3,000

3,000

100

100

r2 = 0.42 r2 = 0.36

A B

Supplementary Figure 8. Scatter plots and Pearson correlation coefficients for (A) Sense, and (B) Anti-sense RPM counts from the exponential phase for all CDS regions in Arnvig et al. and this study.

Arn

vig

et a

l.

Arn

vig

et a

l.

This study This study

Cum

ulat

ive

RP

M

Cum

ulat

ive

RP

M

Genomic PositionGenomic Position

Forward Strand

A B

Supplementary Figure 9. Plot of the cumulative RPM (normalized to 1 for the total sum) for TUs on the forward and reverse strand against TU Start (genomic position in fraction of genome size) in (A) Exponential Phase and (B) Stationary Phase. The two curves are significantly different on a Kolmogorov-Smirnov test (p < 10-10 in both conditions).

Forward Strand

Reverse Strand Reverse Strand

Exponential Phase Stationary Phase