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| Status |
Public on Jan 01, 2019 |
| Title |
4E: replicate C, 5 minutes |
| Sample type |
SRA |
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| Source name |
bacterial cells
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| Organism |
Picosynechococcus sp. PCC 7002 |
| Characteristics |
strain: PCC 7002
timepoint: 5 minutes
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| Treatment protocol |
2 mL of each culture was collected for cell counts via hemocytometer and OD measurements. 40 mL of culture was collected and deposited into a 50 mL conical tube containing 5.0 mL stop solution (10% phenol in ethanol) placed in ice. Rifampicin was added at a concentration of 50mg/mL (in DMSO) and samples were taken as described above at 0.5, 1, 2.5, 5, 7.5, and 10 minutes following addition. Samples were spun down in a Beckman Coulter Avanti J-E Centrifuge at 7,500xg for 10 minutes at 4C. The supernatant was removed and samples were flash frozen in liquid nitrogen and stored at -80C until RNA extraction (1 week). RNA spike-ins were used for normalization and to ensure read counts corresponded to RNA copy number. Six RNA spike-ins from bacteriophage phiX174 were used as in Chen, 2015. DNA templates for in vitro transcription were created by including the T7 promoter sequence in the primer sequence and amplifying phiX174 DNA (New England Biolabs, #N3023S). DNA templates were purified with ethanol precipitation and used to synthesize RNA with the T7 RiboMAX Express Large Scale RNA Production System (Promega, #P1320). RNA was purified with ethanol precipitation and quantified via Nanodrop. RNA spike-ins were combined to obtain a range of copy numbers per cell (gene H: 0.1 copies/cell; gene D: 1 copy/cell; genes F and G: 10 copies/cell; fragment 290: 100 copies/cell; and fragment 190: 1,000 copies/cell).
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| Growth protocol |
The WT strain was maintained on media A+ agar (1.5%) plates. Three pre-cultures of 20 mL A+ media were bubbled with air overnight. The OD730nm was measured and 1 1L pyrex bottles filled with 900 mL media A+ was inoculated with pre-culture for an OD730nm = 0.01. Cultures were bubbled with air for 24 hours before harvesting (OD ~0.1WT PCC 7002 (Pasteur collection) was grown on grown on media A+ (Stevens, 1973) 1.5% agar plates. All liquid cultures were grown at 37C and bubbled with air. A bubble tube containing 21 mL media A+ was inoculated and was grown for 24 hours. The OD730nm was measured and used to inoculate 3 separate bubble tubes containing 21 mL media A+ at an OD730nm = 0.1, which were grown for 24 hours. The OD730nm was measured and 3 separate 1 L bottles containing 900 mL of media A+ were inoculated with pre-culture to achieve an OD 730nm of 0.01. Cultures were bubbled with air at 37C for 22 hours before sampling.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA spike-ins were added to samples immediately before extraction with hot phenol and DNase-treatment as in RNA was extracted using the hot phenol method outlined in Khodursky A, Bernstein J, Peter B, Rhodius V, Wendisch V, Zimmer D. 2003. Escherichia coli spotted double-strand DNA microarrays: RNA extraction, labeling, hybridization, quality control, and data management. Methods Mol Biol 224:61–78.
RNA was submitted to the University of Wisconsin Biotechnology Gene Expression Center for RNA-sequencing. rRNA was removed with a Ribo-Zero Magnetic Kit and the library was created using a TruSeq Stranded Total RNA Library Kit. Samples were run on an Illumina HiSeq 2500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
replicate C, 5 minutes
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| Data processing |
Sequencing files were aligned to the chromsome (NC_010475), pAQ1 (NC_010476), pAQ3 (NC_010477), pAQ4 (NC_010478), pAQ5 (NC_010479), pAQ6 (NC_010480), pAQ7 (NC_010474), and phiX174 (NC_001422) using Bowtie2 (v 2.2.6) and samtools (v 1.2).
Counts for each feature in the general feature format (.gff) files were obtained with the htseq-count script using union mode and stranded set to reverse (HTseq v 0.6.1).
A linear relationship between log RNA spike-in copy number and read counts was only observed for spike-ins added at a ratio of 0.01 – 10 copies/cell, so only these were used for normalization. All PCC 7002 genes were within this range and all samples had a good correlation between log RNA spike-in copy number and read count with all R2 values were between 0.971 and 0.999. The sum of the reads that aligned to the spike-ins was calculated for each sample and used to create a normalization factor to keep the sum of reads that aligned to the spike-ins constant throughout the time course.
Counts for each feature were normalized with the normalization factor created using the sum of the RNA spike-in reads.
The number of reads that aligned to each position (and strand) in the chromosome and plasmids was counted using Integrated Genome Viewer count tool (window size set to 1 base) on the alignment files. These counts were normalized with the normalization factor determined by the RNA spike-in read counts.
Half lives of each gene were calculated by fitting gene read counts from all 21 samples to an exponential decay model. For most genes a poor fit was found when including all timepoints because the decay happened at the earlier part of the timecourse. The number of timepoints included that gave the best fit (highest R-squared value) was used on a per gene basis. The half life was only calculated if the best R-squard fit was > 0.6. If the gene still had at least 50% of the original read counts at the end timepoint it was classified as stable.
Genome_build: NC_010475, NC_010476, NC_010477, NC_010478, NC_010479, NC_010480, NC_010474, NC_001422
Supplementary_files_format_and_content: .csv files contain the number of reads (normalized for decay) that align to each position of the chromosome and plasmids (row number corresponds to position in chromosome/plasmid). The data is stranded so there are separate files for both the plus and minus strand.
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| Submission date |
Jan 13, 2018 |
| Last update date |
Jan 01, 2019 |
| Contact name |
Gina Gordon |
| E-mail(s) |
gcgordon@wisc.edu
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| Organization name |
University of Wisconsin Madison
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| Department |
Chemical and Biological Engineering
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| Lab |
Brian Pfleger Laboratory
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| Street address |
1415 Engineering Dr
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL21381 |
| Series (1) |
| GSE109174 |
Global RNA half lives in cyanobacteria |
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| Relations |
| BioSample |
SAMN08362449 |
| SRA |
SRX3584630 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2934760_minus_4E_norm.csv.gz |
864.8 Kb |
(ftp)(http) |
CSV |
| GSM2934760_minus_4E_pAQ1_norm.csv.gz |
2.1 Kb |
(ftp)(http) |
CSV |
| GSM2934760_minus_4E_pAQ3_norm.csv.gz |
4.3 Kb |
(ftp)(http) |
CSV |
| GSM2934760_minus_4E_pAQ4_norm.csv.gz |
9.8 Kb |
(ftp)(http) |
CSV |
| GSM2934760_minus_4E_pAQ5_norm.csv.gz |
3.9 Kb |
(ftp)(http) |
CSV |
| GSM2934760_minus_4E_pAQ6_norm.csv.gz |
31.6 Kb |
(ftp)(http) |
CSV |
| GSM2934760_minus_4E_pAQ7_norm.csv.gz |
26.9 Kb |
(ftp)(http) |
CSV |
| GSM2934760_plus_4E_norm.csv.gz |
915.8 Kb |
(ftp)(http) |
CSV |
| GSM2934760_plus_4E_pAQ1_norm.csv.gz |
6.4 Kb |
(ftp)(http) |
CSV |
| GSM2934760_plus_4E_pAQ3_norm.csv.gz |
5.4 Kb |
(ftp)(http) |
CSV |
| GSM2934760_plus_4E_pAQ4_norm.csv.gz |
1.4 Kb |
(ftp)(http) |
CSV |
| GSM2934760_plus_4E_pAQ5_norm.csv.gz |
18.2 Kb |
(ftp)(http) |
CSV |
| GSM2934760_plus_4E_pAQ6_norm.csv.gz |
13.0 Kb |
(ftp)(http) |
CSV |
| GSM2934760_plus_4E_pAQ7_norm.csv.gz |
22.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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