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| Status |
Public on Dec 31, 2020 |
| Title |
11-1-E efgA mutant, 40 min after mock treatment |
| Sample type |
SRA |
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| Source name |
Bacterial cells
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| Organism |
Methylorubrum extorquens |
| Characteristics |
strain: PA1
genotype: efgA
treatment: none
time: 40
biological replicate: 2
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| Treatment protocol |
When OD600 of 2 L cultures = 0.2 (~8 hr), 100 mL of each culture was used to harvest “pretreatment” cells for transcriptomic analysis and remaining cultures were divided into 100 mL aliquots in 250 mL flasks (18 hr for wild-type, 16 hr the ΔefgA mutant) and returned to the incubator for 30 min. All flasks were quickly removed from incubator and treated with i) 500 μL of 1 M formaldehyde (5 WT flasks, 4 ΔefgA mutant flasks), ii) 100uL of 50mg/mL kanamycin (4 flasks of each strain), or iii) left untreated (4 flasks of each strain). Flasks were well-sealed with foil and parafilm and returned to the shaker. Time of treatment is designated as t = 0, pretreatment therefore corresponds to -45 min. This growth regimen was executed on 6 consecutive days to obtain all treated samples in biological triplicate.
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| Growth protocol |
From individual colonies, wild-type (CM2730) and the ΔefgA mutant (CM3745) were inoculated into 2 mL MP medium (3.5 mM succinate) and grown at 30 °C with shaking for 32 hr. Cells were subcultured (1/64) into fresh MP media twice: first into 35 mL (3.5 mM succinate, 24 hr incubation) and then into 2 L (15 mM succinate). When OD600 of 2 L cultures = 0.2 (~8 hr), 100 mL of each culture was used to harvest “pretreatment” cells for transcriptomic analysis and remaining cultures were divided into 100 mL aliquots in 250 mL flasks for treatment.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified from cells for each sample using the RNAsnap method [1]. First, cell pellets were resuspended in 500 µl of RNA extraction buffer (18 mM EDTA, 0.025% w/v SDS, 1% v/v 2-mercaptoethanol, 95% v/v formamide) by vortexing. Following incubation at 95°C for 7 min, cell debris was pelleted by centrifugation at 16000 × g for 5 min at room temperature. The resulting supernatant was purified using the Clean & Concentrator kit incorporating the on-column DNase digestion step (Zymo Research). Yields were determined using a Qubit 2.0 fluorometer with the RNA BR assay kit (ThermoFisher). Then, 1.5 µg of purified RNA for each sample was processed using the Ribozero rRNA Removal Kit (Gram-negative bacteria) (Illumina) according to the manufacturer’s instructions except that reaction volumes were reduced by 50%. Depleted RNA samples were ethanol precipitated.
RNA-Seq library preparation from rRNA-depleted samples began by fragmenting them with the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs) for 110 s followed by ethanol precipitation. Fragmented RNA was treated with 10 U of T4 polynucleotide kinase (NEB) in 20 µl reactions containing 1 mM ATP. After another ethanol precipitation, the NEBNext small RNA Library Prep Set for Illumina (Multiplex Compatible) (New England Biolabs) was used to prepare samples according to recommended protocol, except all reaction volumes were reduced 50% and the SR RT primer concentration was reduced by an additional 50% in the RT primer hybridization step. After performing 15 cycles of PCR, samples were ethanol precipitated, and the DNA concentration was determined via the Qubit HS dsDNA assay kit (Thermofisher). For each sample, 200 ng of DNA was run on a 4% EX E-gel (ThermoFisher). DNA with a size of at least ~200 bases was excised and purified using a gel DNA recovery kit (Zymo Research) with elution in 50 µl of nuclease-free water.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
counts.csv
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| Data processing |
Read trimming was performed using Trimmomatic v0.38 in paired-end mode, if applicable, and removing reads trimmed to fewer than 30 bases.
Read alignment was performed using Bowtie2 v4.8.2 with the -k option, in paired-end mode for reads paired after trimming and in single read mode for others
Raw counts were generated using HTSeq v0.11.4 with options to count read pairs or singleton reads that unambiguously overlapped each gene
Normalized read counts were generated using DeSeq2 in R version 3.6.
Genome_build: NC_010172.1
Supplementary_files_format_and_content: CSV
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| Submission date |
Dec 28, 2020 |
| Last update date |
Dec 31, 2020 |
| Contact name |
Jeffrey E. Barrick |
| Organization name |
The University of Texas at Austin
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| Department |
Molecular Biosciences
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| Street address |
2500 Speedway, Stop A4800
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| City |
Austin |
| State/province |
TX |
| ZIP/Postal code |
78712-1639 |
| Country |
USA |
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| Platform ID |
GPL29545 |
| Series (1) |
| GSE163955 |
Global transcriptional response of Methylorubrum extorquens to formaldehyde stress includes both overlapping and unique gene sets in comparison to antibiotic translational inhibition and expands the role of EfgA |
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| Relations |
| BioSample |
SAMN17172633 |
| SRA |
SRX9743902 |
| 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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