"strain: K-12"	"characteristics_ch1.1"
"substr: MG1655"	"characteristics_ch1.2"
"molecule subtype: In vitro synthesized RNase P (rnpB) RNA"	"characteristics_ch1.3"
"library strategy: SPET-seq"	"data_processing.1"
"FastQ files were examined using the FastQC tool. All the relevant SPET-seq data (nascent RNA) analysis and normalization steps were performed using a custom wrapper built on top of the RNA Framework13. Briefly, reads were clipped from 3’ adapter sequences using Cutadapt v1.10, discarding reads shorter than 15 nucleotides. Forward and reverse reads were independently mapped to the RNase P (rnpB) gene using Bowtie v1.1.2, by allowing up to 7 mapping positions to enable mapping to the 7 E. coli rRNA genes (parameters: -n 2 -m 7 -a --best --strata -5 5 [--norc for forward reads, --nofw for reverse reads]). Forward and reverse mapped reads were then re-paired. Using reverse read mapping positions (corresponding to RNA Polymerase positions along gene), forward reads were split into separate SAM files for each transcription intermediate. SAM files were then passed to the rf-count tool of the RNA Framework to generate RT-stop counts (RC) files, that were then normalized using the rf-norm tool."	"data_processing.2"
"Genome_build: U00096.2"	"data_processing.3"
"Supplementary_files_format_and_content: Mapped reads were rescaled to a size of 10 bp, and BEDGraph files were generated using the genomeCoverageBed utility of the BEDTools suite."	"data_processing.4"
"Supplementary_files_format_and_content: DMS-seq data (pooled Total and Ribo- RNA) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format"	"data_processing.5"
"Supplementary_files_format_and_content: SPET-seq data (pooled Total and Ribo- RNA from both replicates) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For each analyzed transcript, 10 files corresponding to SPET-seq data for the 10 transcription deciles are provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format"	"data_processing.6"
"Supplementary_files_format_and_content: SPET-seq data is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. A file corresponding to each transcription intermediate is provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format"	"data_processing.7"
"RNA was purified on RNA Clean & Concentrator™-5 columns following manufacturer’s instructions, and eluted in 6 μl of nuclease-free water. 6 μl of 2X RNA Loading Dye (ThermoScientific, cat. R0641) were added to purified RNA. Both DMS treated and untreated samples were heated at 95°C for 2 minutes, and immediately placed on ice. Samples were resolved on a 10% TBE-Urea polyacrylamide gel, and a gel slice ranging from 50 nt to the full-length product was cut. Gel slices were crushed by centrifugation through a punctured 0.5 ml tube, and resuspended in 500 μl of Diffusion buffer [500 mM Ammonium acetate; 0.05% SDS] supplemented with 60 U SUPERase• In™ RNase Inhibitor, then rotated at 4°C for 16 hours to allow passive diffusion of RNA fragments into buffer. RNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 6 μl nuclease-free water."	"extract_protocol_ch1.1"
"10 pmol of a pre-adenylated (rApp) adapter were ligated to rnpB nascent RNA in a reaction volume of 20 μl, using 400 U T4 RNA Ligase 2, Deletion Mutant in the presence of 20% PEG-8000, by incubation at 25°C for 2 hours. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and RNA was eluted in 5.5 μl nuclease-free water. RNA was heat-denatured at 70°C for 5 minutes, and reverse transcription was carried out in a final volume of 10 μl, in the presence of 0.5 mM dNTPs, 5 pmol of RT primer, 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, and 100 U SuperScript® III Reverse Transcriptase, by incubation at 50°C for 50 minutes. Template RNA was degraded by adding 1 μl of 1 M NaOH, and incubating at 95°C for 5 minutes. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and cDNA was eluted in 6 μl nuclease-free water. cDNA fragments were resolved on a 10% TBE-Urea polyacrylamide gel, and a gel slice corresponding to fragments in the range of 40-300 nt was cut. DNA was recovered by passive diffusion in Diffusion buffer for 16 hours at 37°C with moderate shaking. cDNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 8.25 μl nuclease-free water. 10 pmol of a 5’-phosphorilated adapter were ligated to the 3’-OH of cDNA fragments in a final reaction volume of 25 μl, in the presence of 0.05 mM ATP, 20% PEG-4000, and 100 U CircLigase™ II ssDNA Ligase, by incubation at 60°C for 4 hours, and 68°C for 2 hours. Adapter-ligated cDNA fragments were purified from excess adapter using 1.8 volumes of Agencourt AMPure XP beads, following manufacturer’s instructions. cDNA was eluted in 20 μl of nuclease-free water, and indexed sequencing adapters were introduced by 15 cycles of PCR in the presence of 25 pmol of each primer, and 25 μl NEBNext® High-Fidelity 2X PCR Master Mix."	"extract_protocol_ch1.2"
"Escherichia coli"	"organism_ch1.1"
"rnpB SPET-seq (in vitro)"	"source_name_ch1.1"
"rnpB SPET-seq (in vitro)"	"title.1"
"In vitro transcription reactions were performed in a final volume of 50 µl. Each reaction contained 2 µl E. coli RNA Polymerase Holoenzyme (NEB, cat. M0551S), 5 µl E. coli RNA Polymerase Buffer (10X), 1 µl SUPERase• In™ RNase Inhibitor (Ambion, cat. AM2696), and 500 ng template DNA. Reactions were incubated at 37°C for 5 minutes to allow formation of the RNA Polymerase-DNA binary complex. Transcription was started by addition of 1 µl NTPs (25 mM each), and incubated at 37°C for 5 minutes. Transcription was stopped by addition of 1 µl DNase I (50 U/µl) and Actinomycin D (Sigma Aldrich, cat. A1410, dissolved in DMSO to 5 μg/μl) to a final concentration of 25 ng/μl. Reactions were diluted by addition of 50 μl E. coli RNA Polymerase Buffer 1X. DMS (Sigma Aldrich, cat. D186309) was diluted 1:6 in 100% Ethanol to a final concentration of 1.76 M. Diluted DMS was added to reactions to a final concentration of 100 mM. For control samples, an equal volume of a 1:6 dilution of nuclease-free water in 100% Ethanol was added. Samples were incubated with moderate shaking (800 RPM) at 25°C for 2 minutes, after which reactions were immediately transferred to ice. Two volumes of ice-cold RNA Binding Buffer from RNA Clean & Concentrator™-5 kit (Zymo Research, cat. R1014) supplemented with DTT (Sigma Aldrich, cat. 43815) to a final concentration of 0.7 M, were added to quench DMS, and samples were vigorously vortexed for 10 seconds."	"treatment_protocol_ch1.1"
"OTHER"	"library_strategy.1"
"strain: K-12"	"characteristics_ch1.1"
"substr: MG1655"	"characteristics_ch1.2"
"molecule subtype: In vitro synthesized RNase P (rnpB) RNA"	"characteristics_ch1.3"
"Escherichia coli"	"organism_ch1.1"
"rnpB SPET-seq (in vitro)"	"source_name_ch1.1"
"In vitro transcription reactions were performed in a final volume of 50 µl. Each reaction contained 2 µl E. coli RNA Polymerase Holoenzyme (NEB, cat. M0551S), 5 µl E. coli RNA Polymerase Buffer (10X), 1 µl SUPERase• In™ RNase Inhibitor (Ambion, cat. AM2696), and 500 ng template DNA. Reactions were incubated at 37°C for 5 minutes to allow formation of the RNA Polymerase-DNA binary complex. Transcription was started by addition of 1 µl NTPs (25 mM each), and incubated at 37°C for 5 minutes. Transcription was stopped by addition of 1 µl DNase I (50 U/µl) and Actinomycin D (Sigma Aldrich, cat. A1410, dissolved in DMSO to 5 μg/μl) to a final concentration of 25 ng/μl. Reactions were diluted by addition of 50 μl E. coli RNA Polymerase Buffer 1X. DMS (Sigma Aldrich, cat. D186309) was diluted 1:6 in 100% Ethanol to a final concentration of 1.76 M. Diluted DMS was added to reactions to a final concentration of 100 mM. For control samples, an equal volume of a 1:6 dilution of nuclease-free water in 100% Ethanol was added. Samples were incubated with moderate shaking (800 RPM) at 25°C for 2 minutes, after which reactions were immediately transferred to ice. Two volumes of ice-cold RNA Binding Buffer from RNA Clean & Concentrator™-5 kit (Zymo Research, cat. R1014) supplemented with DTT (Sigma Aldrich, cat. 43815) to a final concentration of 0.7 M, were added to quench DMS, and samples were vigorously vortexed for 10 seconds."	"treatment_protocol_ch1.1"