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| Status |
Public on Apr 01, 2024 |
| Title |
ppk_delta_t60_riff_rep2 |
| Sample type |
SRA |
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| Source name |
cells
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
tissue: cells
genotype: delta-ppk with hfq-mcherry
biological replicate: biological replicate 2
time: time 60 min
treatment: rifampicin treated
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| Treatment protocol |
Samples were collected before rifampicin addition, or at 10, 30 and 60 min after treatment, and were immediately mixed with RNAProtect® Bacteria Reagent (#1018380, Qiagen); for the post-treatment timepoints, a separate untreated control was collected after the same lag time. Cells were pelleted and stored at -80°C. Each sample was also plated for CFUs at the time of harvest in order to provide a normalization factor (used below). Three biological replicates were performed for each genotype/condition combination.
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| Growth protocol |
N-24 WT and Δppk E. coli were supplemented with 150 µg/mL rifampicin (#R3501, Sigma-Aldrich) and incubated at 37°C with 200 rpm shaking.
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| Extracted molecule |
total RNA |
| Extraction protocol |
On ice, each sample pellet was resuspended in 90 µl of 1X TE (10 mM Tris pH 7.0, 1 mM EDTA pH 8.0), briefly centrifuged to remove liquid from tube walls and transferred to 1.5 ml tube, and then transferred to 1.5 ml microfuge tubes. Then 1 µl of Ready-Lyse Lysozyme Solution (LGC Biosearch Technologies) was added to each sample and mixed. Samples were incubated at 30°C for 15 min and returned to ice. Then, 10 µl of Proteinase K (Thermo-Fisher) was added to each sample and mixed. Samples were incubated at 23°C for 15 min, with resuspension every two minutes then returned to ice. EERC RNA spike-in control mix (Ambion) was diluted 1:25 in water as one batch (8.6 µl of EERC, 206.4 µl water).Then, 5 µl of the diluted mix was added to each sample, except for Δppk t=60 (no rifampicin) replicate 1, which received 1.6 µl instead of 5 µl. After vortexing the samples, they were clarified by centrifugation (12,000 r.c.f., 30 sec.) and up to 250 µl of clarified lysate was transferred to a new tube. The samples were cleaned using RNA Clean & Concentrator-96 (Zymo Research) per manufacturer's directions for total RNA clean-up, and eluted with 25 µl of water per sample. Each sample was transferred to tubes that contained 60 µl of water, 10 µl of 10X Baseline Zero DNase buffer, 5 µl of Baseline Zero DNase (LGC Biosearch Technologies), and 2 µl of RNase Inhibitor, Murine (recombinant, NEB) in each tube. Samples were incubated at 37°C for 30 min. The samples were re-cleaned using RNA Clean & Concentrator-96 (Zymo Research) per manufacturer's directions for total RNA clean-up, and eluted with 20 µl water per sample. The concentration of RNA was determined using 5 µl samples and Quantifluor RNA system (Promega) per manufacturer's directions for quantitating RNA in multiwell plates.
Appropriate amounts of nuclease-free water were added to 0.1 µg of RNA per sample to achieve 11 µl of starting material for NEBNext rRNA Depletion Kit (Bacterial, NEB). The kit was used per manufacturer's directions to remove rRNA from the samples. Library preparation was performed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Samples were prepared per kit directions except that 2.5 µl of diluted (1:100) NEBNext Unique Dual Index UMI Adaptors DNA Set 1 was used instead of the NEBNext Adaptor for Illumina, and that Omega Mag-Bind TotalPure NGS Beads were used instead of NEBNext Sample beads for post second-strand synthesis, post adapter ligation, and post PCR clean-up steps. Pooled libraries were subjected to sequencing on a NextSeq 2000 instrument.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
rRNA depleted RNA, umi library
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| Data processing |
Initial read preprocessing, alignment and QC using the IPOD-HR processing pipeline version 2.7.1
overlaps of aligned reads to genes were quantified using the summarizeOverlaps() function of the R package GenomicAlignments (version 1.26.0) running under R 4.0.2, with options “singleEnd = FALSE, inter.feature = FALSE, ignore.strand = TRUE, mode = "IntersectionStrict",fragments = TRUE”.
ead counts for each feature (gen) were then normalized by the total read count for that sample, and then sequencing depths of genomic features further rescaled by the median abundance of ERCC transcripts in that sample (considering only ERCC transcripts with a relative abundance greater than 5*10-4) in order to provide a relative estimate of per-cell transcript abundance
For each gene, we then fitted an exponential decay equation (y=a*exp(-1*t/b), where y is the normalized transcript abundance, a and b are coefficients to be fitted, and t is the time) to the data for each time course, using the scipy.optimize.curve_fit function from scipy 1.8.0, with default optimization settings, and a and b initialized to 0.001 and 20, respectively; a represents a baseline abundance (at t=0) for each transcript, and b is the exponential decay time constant (in minutes).
Assembly: E coli MG1655 gene product set from regulonDB, plus ERCC spikein mix
Supplementary files format and content: halflives_for_geo.csv – contains the results of the exponential decay fits for each gene/condition combination
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| Submission date |
Aug 21, 2023 |
| Last update date |
Apr 01, 2024 |
| Contact name |
Lydia Freddolino |
| E-mail(s) |
lydsf@umich.edu
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| Organization name |
University of Michigan
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| Department |
Biological Chemistry
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| Lab |
Freddolino Lab
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| Street address |
1150 W. Medical Center Dr., MSRB 3 room 3315
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109-0600 |
| Country |
USA |
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| Platform ID |
GPL33080 |
| Series (2) |
| GSE241321 |
Bacterial Stress Bodies – Ancestral Condensates Regulating RNA Turnover and Protein Translation III |
| GSE241322 |
Bacterial Stress Bodies – Ancestral Condensates Regulating RNA Turnover and Protein Translation |
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| Relations |
| BioSample |
SAMN37102598 |
| SRA |
SRX21638128 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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