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| Status |
Public on May 21, 2019 |
| Title |
5GB1C-5G-La-BR2 : Wild-type, lanthanum added, biological replicate 2 |
| Sample type |
SRA |
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| Source name |
exponential phase culture
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| Organism |
Methylotuvimicrobium buryatense |
| Characteristics |
strain: 5GB1C
genotype/variation: Wild-type
tissue: exponential phase culture
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| Treatment protocol |
Wild-type and lanA- cells were growth in biological duplicate in either 0 µM or 30 µM lanthanum, for a total of 8 samples.
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| Growth protocol |
Starter cultures were grown to late logarithmic phase (OD600 ~ 0.8) and a 2% inoculum (v/v) was transferred into 50 mL NMS2 medium, under an atmosphere of 25% methane and 75% air.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Once samples reached OD600 ~ 0.6-0.7, 2.5 mL of a stop solution of 5% (v/v) phenol in ethanol was added to 22.5 mL of culture. Cells were pelleted for 15 min at 4°C, and resuspended in RNA extraction buffer (a 1:3 ratio of 5% cetrimonium brimide in 2.5 M NaCl to 0.1 M phosphate buffer at pH 5.8). An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1 ratio) was added, and sodium dodecyl sulfate and N-lauroylsarcosine sodium salt were each added at 0.5% (v/v). Cells were lysed in a bead beater with 0.1-mm zirconia-silica beads (Biospec Products, Bartlesville, OK, USA) for 4 min, then centrifuged at 14,000 x g for 5 min. The aqueous fraction was then washed with chloroform-isoamyl alcohol (24:1 ratio), and the aqueous fraction from this wash was precipitated overnight with 150 mM sodium acetate (pH 5.5), 1.5 mM MgCl2, and 50% isopropanol at -80°C. Precipitated RNA was harvested at 14,000 x g for 30 min at 4°C, and treated with DNase I (Ambion, Foster City, CA, USA) before purification with the RNeasy minikit and on-column DNase (Qiagen, Hilden, Germany).
cDNA library preparation and RNA sequencing was performed by GENEWIZ using Illumina HiSeq 2x150 (pair ended) reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Alignment was performed using BWA version 0.7.12-r1044 using the BWA-MEM algorithm and 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 0.6.1p1 in the ‘intersection-nonempty’ mode.
Differential abundance analysis was performed with DESeq2 1.2.10 using R 3.3.0.
Genome_build: CP035467
Supplementary_files_format_and_content: *.dat.txt files are two columns: locus tag and read count. At the bottom of the file five lines of summary read alignment statistics are given.
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| Submission date |
Jan 30, 2019 |
| Last update date |
May 24, 2019 |
| Contact name |
Mitchell William Pesesky |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Street address |
616 NE Northlake Place
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98105 |
| Country |
USA |
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| Platform ID |
GPL26131 |
| Series (1) |
| GSE125909 |
A mutagenic screen identifies a TonB-dependent receptor required for the lanthanide metal switch in the Type I methanotroph “Methylotuvimicrobium buryatense” 5GB1C |
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| Relations |
| BioSample |
SAMN10847881 |
| SRA |
SRX5311928 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3584844_5GB1C-5G-La-BR2.summary.dat.txt.gz |
20.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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