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| Status |
Public on Dec 26, 2016 |
| Title |
MB_31_BR2 |
| Sample type |
SRA |
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| Source name |
bacterial transcriptome
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| Organism |
Methylobacter tundripaludum |
| Characteristics |
culture: Methylobacter trundripaludum 31/32
agent: 25% (v/v) methane added
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| Growth protocol |
M. tundripaludum 31/32 was pre-grown in nitrate mineral salt (NMS) medium, 30 µM lanthanum chloride and 25 % methane, 5 % air and 70 % nitrogen in the headspace. Methylotenera strains were pre-grown in the same way but with 0.1% methanol instead. We established co-cultures and controls in duplicates or triplicates in autoclaved vials in NMS medium. Different strains were mixed together in equal starting densities (107 cells per ml). All vials were sealed with rubber stoppers and capped with crimp seals. The headspace was exchanged daily according to the following scheme: (1) Flushing with N2 gas for 30 sec (flow rate > 1.28 L/min), (2) Equalizing pressure, by removing the excess volume of N2 gas with a syringe, (3) Removing 60 ml of headspace and adding back 50 ml of methane and 10 ml of air, corresponding to an initial dissolved O2 concentration of approximately 15 µM. All vials were incubated in a shaker (200 RPM) at 18 °C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from co-cultures and pure cultures grown with and without lanthanum. Cells were grown to an optical density 600 of approximately 0.5 for extracting RNA. We followed the procedure described earlier Chu et al 2016 Journal of Bacteriology with the following exception: we used the MPBio FastPrep® Lysing E matrix tubes. The purified RNA was tested for DNA contamination using 16S PCR. Afterwards, samples were stored at -80°C for subsequent analyses.
RNA was submitted for transcriptomics analysis to GENEWIZ (Seattle, USA). 1 x 50 bp were sequenced in single-read configuration in Rapid Run mode on a HiSeq 2500 System.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
raw reads were aligned to the combined isolate genomes, as appropriate for each sample, using BWA version 0.7.12-r1044 with default parameters
The alignments were post-processed into sorted BAM files with SAMTools version 1.2-232-g87cdc4a
Reads were attributed to open reading frames (ORFs) using the htseq-count tool from the ‘HTSeq’ framework version 0.5.4p5 in the ‘intersection-nonempty’ mode
Differential abundance analysis was performed with DESeq2 1.2.10 using R 3.2.4
Genome_build: Genebank: JQMG00000000, JUHH01000001, JPOH00000000
Supplementary_files_format_and_content: tab-delimited text files include DESeq raw counts for differernt organisms in pure and mixed culture
Supplementary_files_format_and_content: 001_methanotroph_count: MB_31_BR1, First biological replicate of Methylobacter 31/32 pure culture; MB_31_BR2, Second biological replicate of Methylobacter 31/32 pure culture; MIX_31_13_BR1, First biological replicate of Methylobacter 31/32 and Methylotenera #13 co-culture; MIX_31_13_BR2, Second biological replicate of Methylobacter 31/32 and Methylotenera #13 co-culture; MIX_31_JLW8_BR1, First biological replicate of Methylobacter 31/32 and Methylotenera JLW8 co-culture; MIX_31_JLW8_BR2, Second biological replicate of Methylobacter 31/32 and Methylotenera JLW8 co-culture.
Supplementary_files_format_and_content: 001_nonmethanotroph_MT13: MIX_31_JLW8_BR1, First biological replicate of Methylotenera #13 pure culture; MIX_31_JLW8_BR2, Second biological replicate of Methylotenera #13 pure culture; MT_JLW8_BR1, First biological replicate of Methylobacter 31/32 and Methylotenera #13 co-culture; MT_JLW8_BR2, Second biological replicate of Methylobacter 31/32 and Methylotenera #13 co-culture
Supplementary_files_format_and_content: 001_nonmethanotroph_MTJLW8: MIX_31_JLW8_BR1, First biological replicate of Methylotenera JLW8 pure culture; MIX_31_JLW8_BR2, Second biological replicate of Methylotenera JLW8 pure culture; MT_JLW8_BR1, First biological replicate of Methylobacter 31/32 and Methylotenera JLW8 co-culture; MT_JLW8_BR2, Second biological replicate of Methylobacter 31/32 and Methylotenera JLW8 co-culture
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| Submission date |
Aug 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sascha Krause |
| E-mail(s) |
saschak@uw.edu
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| Phone |
2062955279
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| Organization name |
University of Washington
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| Department |
Microbiology
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| Lab |
Lidstrom Lab
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| Street address |
616 NE Northlake Place
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98105 |
| Country |
USA |
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| Platform ID |
GPL22331 |
| Series (1) |
| GSE85736 |
A transcriptomics approach to compare transcriptomes of individual methylotrophs from Lake Washington sediment in artificial communities and in pure cultures. |
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| Relations |
| BioSample |
SAMN05583633 |
| SRA |
SRX2033432 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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