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| Status |
Public on Mar 06, 2021 |
| Title |
ctc DMS |
| Sample type |
SRA |
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| Source name |
planktonic cells
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| Organism |
Bacillus subtilis subsp. subtilis str. 168 |
| Characteristics |
strain: 168
genotype: wild-type
treatment: treated with DMS for 2 min
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| Treatment protocol |
Two cultures were grown in parallel. After subjecting one of the two cultures to DMS (~5% final concentration) for 2 min, both cultures were treated with chilled stop solution (30% β-mercaptoethanol, 25% isoamyl alcohol), washed with chilled wash solution (30% β-mercaptoethanol), and frozen as cell pellets.
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| Growth protocol |
BS168 cultures were grown to exponential phase (OD600≈0.2) in LB liquid media by back-diluting an overnight culture.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Thawed cell pellets were treated with lysozyme and total RNA lysis buffer (10 mM EDTA, 50 mM sodium acetate). Total RNA was extracted using hot acid-phenol:chloroform and isopropanol precipitation.
Libraries were prepared according to established protocols from Tomezsko et al., Nature, 2020 and Zubradt et al., Nature Methods, 2016. In brief, DNA was removed using TURBO DNase, RNA >200 nt was purified using an RNA Clean & Concentrator-5 Kit, ribosomal RNA was depleted using a MICROBExpress Bacterial mRNA Enrichment Kit, and RNA >200 nt was again purified in the same manner. For each sample, gene-specific reverse transcription was performed for ctc and yvrE using TGIRT-III. RNA was removed using RNase H and DNA was PCR amplified for 15-25 cycles. ~250 bp amplicons were purified by gel extraction and isopropanol precipitation. Samples with low DNA concentrations were reamplified for 7-20 additional cycles and purified in the same manner. Illumina adapters were added via PCR and libraries were sequenced on an Illumina MiSeq (2 x 250 nt reads) per the manufacturer’s instructions.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Library strategy: DMS-MaPseq
Built-in Illumina software was used for base calling.
Paired-end reads were filtered for quality and trimmed using FASTQC v.0.11.8 and TrimGalore 0.4.1, respectively. Processed reads were aligned to target sequences in the reference genome NC_000964.3 using Bowtie2 2.3.4.1 with the parameters ‘--local --no-unal --no-discordant --no-mixed -X 1000 -L 12’.
Mapped reads were converted to bit vectors and clustered by their mutational signatures using the DREEM algorithm with established parameters from Tomezsko et al., Nature, 2020.
Genome_build: NC_000964.3
Supplementary_files_format_and_content: Tab-delimited text files contain the population average mutational fractions for each sample. The per-base fractions of mismatches as well as mismatches and deletions were calculated from bit vectors.
Supplementary_files_format_and_content: Tab-delimited text files contain the DNA sequence and DMS signals for each sample. DMS signals were quantified as the mutation rates of the bases in the cluster K=1 following expectation-maximization (EM) clustering.
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| Submission date |
Mar 06, 2021 |
| Last update date |
Mar 07, 2021 |
| Contact name |
Gene-Wei Li |
| E-mail(s) |
gwli@mit.edu
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| Phone |
617-324-6703
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| Organization name |
Massachusetts Institute of Technology
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| Department |
Biology
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| Lab |
Gene-Wei Li Lab
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| Street address |
31 Ames Street, 68-223A
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL23473 |
| Series (1) |
| GSE168393 |
Translational activation by an alternative sigma factor in Bacillus subtilis |
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| Relations |
| BioSample |
SAMN18183188 |
| SRA |
SRX10251081 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5137909_ctc_DMS_K1_Clusters_Mu.txt.gz |
1.0 Kb |
(ftp)(http) |
TXT |
| GSM5137909_ctc_DMS_popavg_reacts.txt.gz |
1.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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