Bacterial strains and growth conditions
Table of Contents
The Mtb H37Rv strain (obtained from C. Sassetti) was grown at 37 °C in a minimal medium (Difco Middlebrook 7H9 broth (BD, 271310) supplemented with 0.5% (v/v) glycerol, 0.05% (v/v) tyloxapol (Sigma, T8761), 0.2 g l−1 casamino acids (BD, 223050), and 10% (v/v) OADC (oleic acid, albumin, dextrose and catalase; BD, 212351)). The double-auxotrophic Mtb mc26206 strain (H37Rv ΔpanCDΔleuCD)42 (obtained from W. Jacobs Jr) was grown in the minimal medium with an additional 50 mg l−1 l-leucine (Sigma, L8000) and 24 mg l−1 pantothenic acid (Sigma, P5155). The Msm mc2155 strain (obtained from S. Fortune) was grown in the Middlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween-80 (VWR, M126), and 10% (v/v) albumin–dextrose–catalase. Liquid Mtb and Msm cultures were grown at 37 °C in Nalgene sterile square PETG medium bottles with constant agitation. The solid Mtb culture was grown on 7H11 agar (Sigma, M0428) supplemented as described above except for tyloxapol.
CRISPR interference
Plasmid pIRL58 (Addgene, 166886) bearing the Streptococcus thermophilus CRISPR–dCas9 system (dCas9Sth1)13 was used to modulate the RNA expression level of target genes in Mtb mc26206 cells. Oligonucleotides for single guide RNAs (sgRNAs; Integrated DNA Technologies) were cloned into pIRL58. After verification by Sanger sequencing, pIRL58 and pIRL19 (Addgene, 163634, which supplied the L5 integrase function on a separate suicide vector) were co-transformed into Mtb cells by electroporation using GenePulser (BioRad) at 2,500 V, 700 Ω, and 25 μF. Single colonies were picked from the solid culture plates with 20 μg ml−1 kanamycin (Goldbio, K-120) selection after 14–21 days of culture. Target gene knockdown was induced by adding 100 ng ml−1 ATc (Sigma, 37919). The sgRNA and primer sequences are listed in Supplementary Table 5.
SEnd-seq
RNA isolation
Bacterial cells were quenched by adding 1× vol of GTC buffer (600 g l−1 guanidium thiocyanate, 5 g l−1 N-laurylsarcosine, 7.1 g l−1 sodium citrate, and 0.7% (v/v) β-mercaptoethanol) to the culture medium immediately before collection and placed at room temperature for 15 min. Cell pellets were collected by centrifugation at 4,000g for 10 min at 4 °C, and then thoroughly resuspended in 100 μl TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). After the addition of 1 ml TRIzol reagent (Invitrogen, 15596) and 350 mg of glass beads (Sigma, G1145), the cells were immediately lysed in a screw-cap tube by bead beating with the Precellys Evolution homogenizer (Bertin Technologies, 02520-300-RD000) at 10,000 r.p.m. for 4× 45-s cycles with a 60-s interval and chilled with dry ice. After removal of the beads by spinning samples at 12,000 r.p.m. for 5 min at 4 °C, the liquid phase was transferred to a new tube. A 200 μl volume of chloroform was added, and the sample was gently inverted several times until reaching homogeneity. The sample was then incubated for 15 min at room temperature before spinning at 12,000g for 10 min at 4 °C. The upper phase (about 600 μl) was gently collected and mixed at a 1:1 ratio with 100% isopropanol. The sample was incubated for 2 h at −20 °C and then centrifuged at 14,000 r.p.m. for 15 min at 4 °C. The pellet was washed twice with 1 ml of 75% (v/v) ethanol, air dried for 5 min and dissolved in nuclease-free water. RNA integrity was assessed with 1% (m/v) agarose gel and the Agilent 2100 Bioanalyzer System (Agilent Technologies, 5067-4626). For antibiotic treatment conditions, Mtb mc26206 cells were exponentially grown to an optical density at 600 nm (OD600) of about 0.8 followed by treatment with a specific antibiotic (30 μg ml−1 linezolid (Sigma, PZ0014), 40 μg ml−1 clarithromycin (Sigma, C9742), 300 μg ml−1 streptomycin (Sigma, S9137), or 50 μg ml−1 rifampicin (Sigma, R3501)). At each time point following the treatment, 4 ml of cell culture medium was withdrawn and mixed quickly with 4 ml GTC buffer. The cells were then collected, and the RNA was isolated as described above.
Library preparation for total RNA SEnd-seq
A 5 μg quantity of total RNA was mixed with pooled spike-in RNAs used in our previous study3 at a mass ratio of 300:1 in a total volume of 12 μl. The RNA sample was incubated with a 5′-adaptor ligation mix (1 μl of 100 μM 5′ adaptor (Supplementary Table 5), 0.5 μl of 50 mM ATP, 2 μl DMSO, 5 μl of 50% PEG8000, 1 μl RNase Inhibitor (New England BioLabs, M0314), and 1 μl of High Concentration T4 RNA Ligase 1 (New England BioLabs, M0437)) at 23 °C for 5 h. The sample was then diluted to 40 μl with nuclease-free water and cleaned twice with 1.5× vol of Agencourt RNAClean XP beads (Beckman Coulter, A63987). Immediately following the 5′ adaptor ligation, the eluted RNA was ligated to the 3′ adaptor (Supplementary Table 5) using the same procedure. After incubation at 23 °C for 5 h, the reaction was diluted to 40 μl with water and cleaned twice with 1.5× vol of Agencourt RNAClean XP beads to remove excess adaptors. The sample was subsequently eluted with 0.1× TE buffer and subjected to ribosomal RNA removal with RiboMinus Transcriptome Isolation Kit (Thermo Fisher, K155004) following the manufacturer’s instructions. After recovery by ethanol precipitation, the RNA was reverse transcribed to cDNA with Eubacterium rectale maturase (recombinantly purified from Eco, obtained from A. M. Pyle)43 and 5′-phosphorylated and biotinylated reverse transcription primer (Supplementary Table 5). After purification, the cDNA was circularized with TS2126 RNA ligase44 (obtained from K. Ryan). Double-stranded DNA was synthesized by DNA PolI (New England BioLabs, M0209S). After enzyme inactivation and DNA purification with 1.5× vol of AMPure beads (Beckman Coulter, A63882), the DNA was subsequently fragmented by dsDNA Fragmentase (New England BioLabs, M0348S) at 37 °C for 15 min. The reaction was stopped by adding 5 μl of 0.5 M EDTA and incubated at 65 °C for 15 min in the presence of 50 mM dithiothreitol (DTT). Next, the DNA was diluted to 40 μl with TE buffer and purified with 1.5× vol of AMPure beads. The eluted DNA was used for sequencing library preparation with NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, E7645). Biotinylated DNA fragments were enriched by 5 μl of Dynabeads M-280 Streptavidin (Thermo Fisher, 11205D) and further amplified for 12 cycles by PCR.
Library preparation for primary RNA SEnd-seq
A 5 μg quantity of total RNA was used for primary transcript enrichment with our previously published method3. In brief, the 5′-triphosphorylated RNA species was specifically capped with 3′-desthiobiotin-GTP (New England BioLabs, N0761) by the Vaccinia Capping System (New England BioLabs, M2080S). The RNA was subjected to 3′ adaptor ligation using the same procedure as described above and subsequently enriched with Hydrophilic Streptavidin Magnetic Beads (New England BioLabs, S1421). After washing thoroughly, the RNA was eluted and reverse transcribed to cDNA as described above. The remaining steps were the same as those for library preparation for total RNA SEnd-seq, except that the DNA library was amplified for 15 cycles.
Illumina sequencing
Following PCR amplification, each amplicon was cleaned by 1× vol of AMPure XP beads twice and quantified with a Qubit 2.0 fluorometer (Invitrogen). The amplicon size and purity were further evaluated on an Agilent 2200 Tape Station (Agilent Technologies, 5067-5576). Equal amounts of amplicon were then multiplexed and sequenced with 2 × 150 cycles on an Illumina NextSeq500 or NovaSeq6000 platform (Rockefeller University Genomics Resource Center).
SEnd-seq data analysis
Data processing
After quality filtering and Illumina sequencing adaptor trimming with FASTX-Toolkit (v0.0.13), the raw paired-end reads were merged to single-end reads by using FLASh software (v1.2.11). The correlated 5′-end and 3′-end sequences were extracted by the custom script (fasta_to_paired.sh) using the SeqKit (v2.4.0) and Cutadapt (v4.1) packages. The inferred full-length reads were generated by Bedtools (v2.31.0) and Samtools (v1.17) after mapping to the reference genome (NC_000913.3 for Eco, NC_008596.1 for Msm and NC_018143.2 for Mtb) with Bowtie 2 (v2.5.1). The full-length reads with an insert length greater than 10,000 nt were discarded. The mapping results were visualized using the IGV genome viewer (v2.4.10). Data analysis and visualization scripts used Python packages including Matplotlib (v3.7.1), Numpy (v1.24.3), Scipy (v1.10.1), bioinfokit (v0.3), and pyCircos (v0.3.0).
RNA coverage
Each full-length read was first mapped to the genome in a specific direction. Directional RNA coverage was quantified by summing the number of aligned reads at each mapped nucleotide position. When comparing RNA coverage between samples, data were normalized by the total non-ribosomal RNA amount in each sample. For the samples treated with translation inhibitors, the abundance of spike-in RNAs was used for normalization. Coding TUs and asRNAs with high levels of expression were defined as those with an average RNA coverage of at least 10 for the first 100 nt downstream of the TSS. The circos plot was generated using the Python package pyCircos (github.com/ponnhide/pyCircos, version 0.2.0). The RNA coverage plots were generated using Matplotlib package45 and custom Python scripts.
TSS identification
TSSs were identified from the primary RNA SEnd-seq data using a custom Python script. Only positions with more than 10 reads starting at that position, and with an increase of at least 50% in read coverage compared to its upstream neighbouring position (for example, 50 reads at position −1 and 150 reads at position 0), were retained. Candidate TSS positions within 5 nt in the same orientation were grouped together, and the position with the largest amount of read increase was used as the representative TSS position. Motif analysis around the TSS regions (−40 nt to +5 nt) was carried out by MEME (v5.5.2)46.
TTS identification
Potential TTSs were identified from the total RNA SEnd-seq data at genomic positions with more than 10 reads ending at that position (outside rRNA genes) and with a reduction of more than 40% in read coverage compared to its upstream neighbouring position (for example, 100 reads at position −1 and 50 reads at position 0).
TU annotation
TUs were used in this work to analyse the transcription of coding genes. The genome was first segmented into preliminary TUs that contained annotated genes of the same direction. A preliminary unit was further segmented into multiple units if it contained any internal TSS with a strong activity (>2-fold increase in RNA coverage between downstream and upstream of the site for log-phase cell sample). As such, each TU contains a major TSS (TU start site) and possibly additional minor TSSs (<2-fold increase in RNA coverage). The end site of a TU was set to 10 nt before the start of a following co-directional TU, or the middle position between opposite genes that belong to two convergent TUs. TUs shorter than 700 nt and TUs annotated with only rRNA or tRNA genes were excluded from further analysis.
Antisense transcript annotation
asRNAs were called if there existed a strong antisense TSS within a given coding TU or if an opposite-direction TSS was found within the non-annotated 400-nt region downstream of a coding TU. The end site of an asRNA was set to the position where the RNA coverage dropped below 25% of the peak value.
PF analysis
Each coding TU was assigned with an upstream zone (from 0 to 200 nt downstream of the TSS) and a downstream zone (from 500 nt downstream of the TSS to the end of the TU). If there was another qualified TSS located within the downstream zone, the region downstream of that TSS was excluded from analysis. The ratio between the average RNA intensity of the downstream zone and that of the upstream zone was calculated as the PF for the corresponding TU. For asRNAs, the upstream and downstream zones were defined as 0–200 nt and 500–700 nt downstream of the TSS, respectively. The lower and upper bounds of PF values were set to be 0.0 and 2.0, respectively.
Gene ontology analysis
The Database for Annotation, Visualization, and Integrated Discovery (DAVID; v2023q2; https://david.ncifcrf.gov/)47 was used to carry out gene ontology analysis for Mtb genes with different PF values. The complete list of genes within each set was uploaded to DAVID under the headings of Cellular Compartment, Biological Process, and Molecular Function. Enriched categories with a P value < 0.05 were presented.
NET-SEnd-seq
Cell collection, lysis, and elongation complex pulldown protocols were adapted from a published study16 with modifications. Briefly, an ATc-inducible pIRL58 backbone plasmid bearing Mtb rpoC–6×His was transformed into Mtb mc2 6206 cells and the genome-integrated expression strain was picked as described above. For each pulldown sample, 55 ml of cell culture was prepared. When the cell culture reached the mid-log phase (OD600 = 0.5), 100 ng ml−1 ATc or an equivalent volume of solvent methanol was added to the medium, and the cells were cultured for another 12 h. After removing 4 ml of cell culture for total RNA extraction, the remaining cell culture was mixed with an equal volume of frozen 2× crush buffer (20 mM Tris-HCl pH 7.8, 10 mM EDTA, 100 mM NaCl, 1 M urea, 25 mM NaN3, 2 mM β-mercaptoethanol, 10% ethanol, 0.4% NP40, and 1 mM phenylmethylsulfonyl fluoride). The cells were subsequently precipitated by centrifugation at 4,000g for 10 min at 4 °C, immediately frozen in liquid nitrogen, and stored at −80 °C for at least 1 day. After thawing on ice, the cells were washed twice with 25 ml of cold PBS pH 7.4 and once with 5 ml of cold lysis buffer (20 mM KOH-HEPES pH 7.9, 50 mM KCl, 0.5 mM DTT, 5 mM CaCl2, 10% glycerol, 0.3 mM MgCl2, and 2.5 mM imidazole). The cells were then resuspended in 2 ml of lysis buffer, transferred to two 2-ml lysing matrix B tubes (MP Biomedicals, 116911050), and immediately lysed by bead beating with the Precellys Evolution homogenizer at 10,000 r.p.m. for 4× 45-s cycles with 60-s interval and chilled with dry ice. After centrifugation at 13,000g for 5 min, the supernatant was collected into a new 15-ml RNase-free tube. Each lysing matrix B tube was subjected to an additional round of bead beating with 1 ml of fresh lysis buffer and the supernatants were combined. Next, the collected sample was treated with 1 μl TURBO DNase (Life Technologies, AM2238) and incubated at room temperature for 10 min. After centrifugation at 4,000g for 10 min at 4 °C, the supernatant was transferred to a new 15-ml tube and incubated with 40 μl pre-washed Ni-NTA beads (Qiagen, 30230) for 1 h at 4 °C with continuous shaking at 100 r.p.m. After immobilization, the beads were washed four times with 5 ml of wash buffer (20 mM Tris-HCl pH 7.8, 1 M betaine, 5% glycerol, 2 mM β-mercaptoethanol, and 2.5 mM imidazole) and five times with 5 ml of pre-elution buffer (20 mM Tris-HCl pH 7.8, 40 mM KCl, 5% glycerol, 2 mM β-mercaptoethanol, and 2.5 mM imidazole). The immobilized complex was subsequently eluted with 300 μl of the pre-elution buffer containing 0.3 M imidazole. The nucleic acids in the eluates were extracted once with 200 μl phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and once with 200 µl chloroform. The top aqueous phase was collected and precipitated by 3× volumes of ethanol, 0.1× vol of 3 M sodium acetate pH 5.2, and 2 μl glycogen (Thermo Fisher, AM9510). After precipitation at −20 °C overnight and maximum-speed centrifugation for 20 min, the pellet was washed twice with 300 µl of 75% ethanol. The pellet was then dissolved in 50 µl nuclease-free water and treated with 0.5 U Turbo DNase at 37 °C for 15 min. The residual RNA was extracted by phenol/chloroform/isoamyl alcohol, precipitated by ethanol and recovered in 11.5 µl of nuclease-free water. A 1 µl volume of spike-in RNA was added to each RNA sample, and the RNAs were ligated to a 3′ adaptor. The remaining steps were the same as those described above for total RNA SEnd-seq. The DNA library was amplified for 16 cycles by PCR.
ChIP–seq
A 50 ml volume of mid-log phase Mtb cells (OD600 = 0.8–1.0) were treated with 1% formaldehyde while the culture was agitated at room temperature for 30 min. Crosslinking was quenched by adding glycine to a final concentration of 250 mM for another 30 min while stirring at room temperature. The cells were pelleted by centrifugation at 4,000g for 10 min at 4 °C and washed three times with 20 ml of cold PBS and 0.1× protease inhibitor (Sigma, P8465). The cell pellet was stored at −80 °C for at least one day. After thawing on ice, the cells were washed once with 5 ml of IP lysis buffer (20 mM KOH-HEPES pH 7.9, 50 mM KCl, 0.5 mM DTT, 5 mM CaCl2, and 10% glycerol) and resuspended in 2 ml of IP lysis buffer. The cells were then transferred to two 2-ml lysing matrix B tubes (MP Biomedicals, 116911050) and immediately lysed by bead beating with the Precellys Evolution homogenizer at 10,000 r.p.m. for 4× 45-s cycles with a 60-s interval and chilled with dry ice. After centrifugation at 13,000g for 5 min, the supernatant was collected into a new 15-ml RNase-free tube. Each lysing matrix B tube was subjected to an additional round of bead beating after adding 1 ml of fresh IP lysis buffer. After centrifugation at 4,000g for 10 min at 4 °C and sampling for input control, 4 ml of supernatant was transferred to a new 15-ml tube and incubated with 0.75 µl of micrococcal nuclease (New England BioLabs, M0247S) at 37 °C for 15 min with continuous shaking. The reaction was stopped by adding EDTA at a final concentration of 25 mM, and the supernatant was transferred to a new 15-ml tube after centrifugation at 4,000g for 10 min at 4 °C. A 3 µl volume of anti-Eco σ70-factor antibody (BioLegend, 663208; 1:1,333 dilution) or 5 µl of anti-Eco RNAP β-subunit antibody (BioLegend, 663903; 1:800 dilution) was used to immunoprecipitate Mtb σA-factor and Mtb RNAP, respectively. After overnight incubation, 40 µl of pre-washed protein A/G agarose beads (Thermo Fisher, 26159) were added and incubated for 2 h at 4 °C and for another 30 min at room temperature. The beads were then washed ten times with 5 ml IPP150 buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% NP40) and once with 5 ml TE buffer. Next, the DNA was eluted with 150 µl of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, and 1% SDS) followed by 100 µl TE buffer with 1% SDS. After thoroughly removing the beads by centrifugation at 2,000g for 5 min at 4 °C, the combined supernatants were incubated with 1 mg ml−1 Pronase (Sigma, 537088) at 42 °C for 2 h and then at 65 °C for 9 h. The sample was cleaned twice with 200 µl of phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and recovered by ethanol precipitation. Finally, the sequencing libraries for immunoprecipitated DNA and input control were prepared using the NEBNext Ultra II DNA Library Prep Kit. After sequencing and quality filtering, the reads were mapped to the Mtb genome using Bowtie 2. The ChIP–seq signals were extracted and plotted using custom Python scripts.
Analysis of deposited RNA-seq data
The RNA-seq datasets SRR5689224 and SRR5689225 (BioProject PRJNA390669)12 from log-phase Mtb cells cultured in dextrose-containing medium were used to compare the RNA coverage between SEnd-seq and RNA-seq. The RNA-seq datasets SRR5061507, SRR5061514, SRR5061706 and SRR5061510 (BioProject PRJNA354066)18 from Mtb cells with Rho depletion were used to compare to the rho-knockdown SEnd-seq datasets. The deposited datasets were downloaded from the National Center for Biotechnology Information. After read extraction and quality filtering, the reads were mapped to the Mtb genome using Bowtie 2 (v2.5.1). The RNA intensities were extracted and plotted using custom Python scripts.
Analysis of deposited Ribo-seq data
Mtb Ribo-seq data were downloaded from the EMBL-EBI database (E-MTAB-8835)21. After read extraction and quality filtering, the reads were mapped to the Mtb genome using Bowtie 2. The directional ribosome binding signals were extracted and plotted using a custom Python script.
Immunoblot
Mtb cells were lysed with TRIzol reagent as described above, and protein samples were extracted following a TRIzol-based protein extraction protocol provided by the manufacturer. Immunoblotting was carried out as described previously48. Antibodies against His-tag (Santa Cruz, sc-8036; 1:1,000 dilution), Mtb Rho (obtained from D. Schnappinger; 1:200 dilution), and Eco RpoB (BioLegend, 663903; 1:1,000 dilution) were used.
qPCR
A 1–10 μg amount of total RNA was treated with 0.5 μl of TURBO DNase (Life Technologies, AM2238) at 37 °C for 30 min to remove the genomic DNA. The sample was diluted to 100 μl with RNase-free water and then cleaned three times with 100 μl of H2O-saturated phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v). After ethanol precipitation, 1 μg of RNA was reverse transcribed to cDNA with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, 4368814) following the manufacturer’s instructions. qPCR was conducted using synthesized primers and the SYBR green master mix (Thermo Fisher, 4309155) on a QuantStudio5 Real-Time PCR System (Thermo Fisher). The relative RNA abundance was presented as the signal ratio between the target transcript and the reference 16S rRNA from the same sample using the formula: 2Ct(16S) − Ct(target), in which Ct denotes the cycle threshold.
Inducible lacZ transcription in Mtb
Plasmid pIRL58 was modified by removing the sgRNA expression cassette and replacing the dCas9Sth1 gene body with the Eco lacZ coding region, allowing the synthesis of lacZ RNA under the control of ATc-inducible promoter Ptet. The modified plasmid was co-transformed into Mtb mc26206 cells with pIRL19 as described above. Cells from a single colony of Mtb Ptet-lacZ after selection were exponentially grown to an OD600 of about 0.8 followed by the addition of 100 ng ml−1 ATc to induce lacZ transcription. After induction, 4 ml of cell culture was withdrawn at indicated time points and mixed with 4 ml GTC buffer in a new tube as sample t (St). One extra sample taken immediately before ATc addition was referred to as S0. After RNA isolation and TURBO DNase treatment as described above, 1 µg of total RNA was used to synthesize the cDNA for qPCR. The relative lacZ mRNA abundance at each time point is defined as 2Ct(S0) − Ct(St), in which Ct denotes the cycle threshold.
In vitro transcription
DNA fragments were amplified by PCR from Mtb genomic DNA with primer sets listed in Supplementary Table 5. An AP3 promoter sequence was inserted into one end of the fragment and an intrinsic terminator (derived from TsynB in pIRL58) was placed at the other end. The DNA fragment was then incorporated into the pUC19 plasmid. The plasmid templates were prepared from Eco DH5α cells and subsequently treated with 2 µl RNase A (Thermo Fisher, EN0531) for 30 min and 2 µl Proteinase K (New England BioLabs, P8107S) for 1 h. The plasmid templates were cleaned three times with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and recovered by ethanol precipitation.
To prepare templates with a preformed bubble, the DNA fragment containing the intrinsic terminator was amplified from the plasmid DNA described above by PCR. The product was cleaned with QIAQuick PCR purification kit (Qiagen, 28104) and phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v). The bubble template was constructed by ligating a DNA adaptor (NEBNext adaptor for Illumina) to each end of the DNA fragment using NEBNext Ultra II DNA Library Prep Kit. After XbaI digestion (cut site immediately after the terminator), the DNA template was purified using AMPure XP beads.
Purified Mtb RNAP, σA-factor, NusA, and NusG were prepared as described previously38,49,50. The in vitro transcription mixture contained 2 μl of 10× transcription buffer (200 mM Tris-acetate pH 7.9, 0.5 M potassium acetate, 100 mM magnesium acetate, 10 mM DTT, and 50 µg ml−1 BSA), 1 μl RNase inhibitor, 0.5 pmol of DNA template, and 2 pmol of Mtb RNAP holoenzyme (or core RNAP alone) in a 20 μl volume. The mixture was incubated at 37 °C for 15 min before the addition of rNTPs (100 μM each). At indicated time points, the reaction was quenched by adding EDTA at a final concentration of 20 mM and 2 μl of Proteinase K and incubating for 30 min. The reaction was then diluted to 100 μl with RNase-free H2O and cleaned three times with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v). After ethanol precipitation and resuspension with 30 μl RNase-free H2O, 0.5 μl DNase I (New England BioLabs, M0303S), 3.5 μl of DNase buffer, and 1 μl RNase inhibitor were added. After incubation at 37 °C for 30 min, the RNA product was cleaned three times with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and recovered by ethanol precipitation. Half of the RNA was converted to cDNA with the High-Capacity cDNA Reverse Transcription Kit and evaluated by qPCR as described above. RNA abundances were normalized to a diluted plasmid DNA sample with a concentration of 0.033 ng ml−1.
Statistics
Statistical analyses were conducted with Excel (version 16.178.3) or GraphPad Prism (version 10.1.0). GraphPad Prism (version 10.1.0) or the Python Matplotlib package (version 3.7.1) was used for plotting.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.