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| Status |
Public on Oct 03, 2024 |
| Title |
WT,Stress_CAN,Slevel_HIGH,T24,REPLICATE_4 |
| Sample type |
SRA |
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| Source name |
WT RB-TN Library
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| Organism |
Caulobacter vibrioides |
| Characteristics |
cell line: WT RB-TN Library
cell type: transposon mutagenesis library
genotype: WT
treatment: Stress_CAN,Slevel_HIGH
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| Treatment protocol |
Control condition: Libraries were back diluted to OD 0.008 into 7 ml of PYE or PYE+0.002% xylose and grown overnight until they reached saturation at OD ∼1.6.
Heat stress: Libraries diluted to OD of 1 and heat-stressed at low, medium, or high (37, 42, 43.8◦C, respectively) for 45 minutes in a Biorad Thermocycler. After 45 minutes, cells diluted back to a final OD of 0.008 in 7 ml media for 24-hour growth.
Oxidative stress: Libraries were directly diluted back to OD of 0.008 in 7 ml media that contains low, medium, or high (0.025mM, 0.05mM, 0.1mM) level hydrogen peroxide. Cells were grown for 24 hours in these chronic stress conditions.
L-canavanine stress: Libraries were directly diluted back to OD of 0.008 in 7 ml media that contains low, medium, or high (25ug/ml, 50ug/ml, 100ug/ml) levels of L-canavanine. Cells were grown for 24 hours in these chronic stress conditions.
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| Growth protocol |
Transposon mutagenesis libraries were thawed and recovered in PYE or PYE + %0.2 Xylose overnight at saturating concentrations to minimize growth during recovery. Only for dnaK-NI library, overnights were recovered in saturating Xylose concentrations (%0.2), and treatment and T24 control conditions were grown in minimal Xylose concentrations. (%0.002)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following overnight growth, 1 ml of saturated culture from each Tn library was pelleted at 8000xg for 2 minutes. Genomic DNA was extracted by Monarch Genomics DNA Preparation Kit (NEB) according to the manufacturer’s protocol.
The sequencing libraries for Next-generation sequencing were prepared using a customized three-step PCR protocol. Genomic DNA input was normalized to a concentration of 100 ng/μl. The initial transposon junction amplification (PCR1) was carried out using an arbitrary PCR amplification method. This step utilized a forward primer designed to align with one end of the transposon and three reverse arbitrary primers. The PCR1 process employed a 2-step cycling protocol with annealing temperatures set at 42°C and 58°C, and number of cycles at 6 and 15, respectively. Subsequently, the second PCR step (PCR2) involved the addition of 16S adapters for Illumina indexing, along with unique molecular identifiers (UMI), and amplification of the library for 36 cycles. Post-PCR2 cleanup was performed using Aline PCRClean DX magnetic beads. The final PCR step (PCR3) incorporated NexteraXT indexing in accordance with the manufacturer's protocol. Post-indexed library (PCR3) cleanup was carried out using Aline PCRClean DX magnetic beads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Samples were de-multiplexed, and unique molecular identifiers (UMIs) were added during PCR steps removed using Je
The clipped reads were mapped to the Caulobacter crescentus NA1000 genome (NCBI Reference Sequence: NC011916.1) using bwa and sorted with samtools.
Duplicate transposon reads removed by Je and indexed with samtools.
Genome positions are assigned to the 5′ position of transposon insertions using bedtools genomecov.
bedtools map is used to count either the total number of transposon insertions per gene using the bedtools map -o sum argument or the unique number of insertions using the bedtools map -o count argument
Assembly: NCBI Reference Sequence: NC011916.1
Supplementary files format and content: tab delimited text files include raw total or unique insertion counts for each gene.
Library strategy: Tn-seq
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| Submission date |
Oct 03, 2023 |
| Last update date |
Oct 03, 2024 |
| Contact name |
Peter Chien |
| E-mail(s) |
[email protected]
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| Organization name |
University of Massachusetts Amherst
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| Department |
Biochemistry and Molecular Biology
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| Lab |
Peter Chien
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| Street address |
240 Thatcher Rd, Life Science Laboratories Room N325
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| City |
Amherst |
| State/province |
MA |
| ZIP/Postal code |
01003 |
| Country |
USA |
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| Platform ID |
GPL24555 |
| Series (1) |
| GSE244581 |
Discovering Genetic Modulators of the Protein Homeostasis System through Multilevel Analysis |
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| Relations |
| BioSample |
SAMN37667930 |
| SRA |
SRX21982065 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7821112_BA10-8_S8_R1_001.totalcounts.tab.gz |
16.9 Kb |
(ftp)(http) |
TAB |
| GSM7821112_BA10-8_S8_R1_001.uniquecounts.tab.gz |
14.4 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
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