ERX5974874 E-MTAB-10777:168_RNAseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166634 SAMEA9441794 168_RNAseq_a_s RNA-Seq TRANSCRIPTOMIC size fractionation Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesiswas performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (16 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 Experimental Factor: protocol RNA-seq ERX5974875 E-MTAB-10777:168_RNAseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166635 SAMEA9441779 168_RNAseq_b_s RNA-Seq TRANSCRIPTOMIC size fractionation Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesiswas performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (16 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 Experimental Factor: protocol RNA-seq ERX5974876 E-MTAB-10777:168_rpoD_cappableseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166636 SAMEA9441780 168_rpoD_cappableseq_a_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. At exponential phase, cultures were induced with 3mM IPTG (final) for 1 hour before harvesting cells as described for other samples. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 amyE::Phyperspank - rpoD (spec) Experimental Factor: protocol Cappable-seq ERX5974877 E-MTAB-10777:168_rpoD_cappableseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166637 SAMEA9441781 168_rpoD_cappableseq_b_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. At exponential phase, cultures were induced with 3mM IPTG (final) for 1 hour before harvesting cells as described for other samples. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 amyE::Phyperspank - rpoD (spec) Experimental Factor: protocol Cappable-seq ERX5974878 E-MTAB-10777:?rok_cappableseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166638 SAMEA9441782 Δrok_cappableseq_a_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 Δrok::kan Experimental Factor: protocol Cappable-seq ERX5974879 E-MTAB-10777:?rok_cappableseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166639 SAMEA9441783 Δrok_cappableseq_b_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 Δrok::kan Experimental Factor: protocol Cappable-seq ERX5974880 E-MTAB-10777:?rok_RNAseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166640 SAMEA9441784 Δrok_RNAseq_a_s RNA-Seq TRANSCRIPTOMIC size fractionation Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesiswas performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (16 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 Δrok::kan Experimental Factor: protocol RNA-seq ERX5974881 E-MTAB-10777:?rok_RNAseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166641 SAMEA9441785 Δrok_RNAseq_b_s RNA-Seq TRANSCRIPTOMIC size fractionation Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesiswas performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (16 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 Δrok::kan Experimental Factor: protocol RNA-seq ERX5974882 E-MTAB-10777:?rpoE_cappableseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166642 SAMEA9441786 ΔrpoE_cappableseq_a_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 ΔrpoE::kan Experimental Factor: protocol Cappable-seq ERX5974883 E-MTAB-10777:?rpoE_cappableseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166643 SAMEA9441787 ΔrpoE_cappableseq_b_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017). Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain 168ca Experimental Factor: genotype trpC2 ΔrpoE::kan Experimental Factor: protocol Cappable-seq ERX5974884 E-MTAB-10777:MG1655_cappableseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166644 SAMEA9441788 MG1655_cappableseq_a_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Experimental Factor: protocol Cappable-seq ERX5974885 E-MTAB-10777:MG1655_cappableseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166645 SAMEA9441789 MG1655_cappableseq_b_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Experimental Factor: protocol Cappable-seq ERX5974886 E-MTAB-10777:?hns_cappableseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166646 SAMEA9441790 Δhns_cappableseq_a_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Δhns::kanR Experimental Factor: protocol Cappable-seq ERX5974887 E-MTAB-10777:?hns_cappableseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166647 SAMEA9441791 Δhns_cappableseq_b_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Δhns::kanR Experimental Factor: protocol Cappable-seq ERX5974888 E-MTAB-10777:?hns_RNAseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166648 SAMEA9441792 Δhns_RNAseq_a_s RNA-Seq TRANSCRIPTOMIC size fractionation Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesiswas performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (16 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Δhns::kanR Experimental Factor: protocol RNA-seq ERX5974889 E-MTAB-10777:?hns_RNAseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166649 SAMEA9441793 Δhns_RNAseq_b_s RNA-Seq TRANSCRIPTOMIC size fractionation Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesiswas performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (16 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Δhns::kanR Experimental Factor: protocol RNA-seq ERX5974890 E-MTAB-10777:?hns_phns-rok_cappableseq_a_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166650 SAMEA9441796 Δhns_phns-rok_cappableseq_a_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Δhns::kanR puc19/Phns-rok Experimental Factor: protocol Cappable-seq ERX5974891 E-MTAB-10777:?hns_phns-rok_cappableseq_b_s ERP130695 PRJEB46508 Different xenogeneic silencing strategies in Escherichia coli and Bacillus subtilis are dictated by RNA polymerase promiscuity ERS7166651 SAMEA9441795 Δhns_phns-rok_cappableseq_b_s RNA-Seq TRANSCRIPTOMIC other Duplicate cultures of strains were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen. For E. coli cells, total RNA was extracted as described in Warman, E.A., Forrest, D., Guest, T. et al. Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol 6, 746–756 (2021). https://doi.org/10.1038/s41564-021-00898-9 Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016). NextSeq 500 Experimental Factor: strain MG1655 Experimental Factor: genotype K-12 F– λ– ilvG– rfb-50 rph-1 Δhns::kanR puc19/Phns-rok Experimental Factor: protocol Cappable-seq