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| Status |
Public on Oct 15, 2014 |
| Title |
t=100 |
| Sample type |
SRA |
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| Source name |
Total RNA lystate
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| Organism |
Caulobacter vibrioides NA1000 |
| Characteristics |
strain: NA1000
timepoint (minutes): 100
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| Treatment protocol |
Cells were pelleted and immediately frozen in liquid nitrogen
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| Growth protocol |
Caulobacter crescentus NA1000 were grown to mid-log (OD600 0.4) at 28C in minimal media M2G (Ely, B. Methods in Enzymology 1991). Cells were then syncrhonized using ludox density centrifugation (Evinger, M., Agabian, N. J. Bacteriol. 1977) and cell cycle time points were collected in 20 minute intervals.
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| Extracted molecule |
total RNA |
| Extraction protocol |
10 micogram of total RNA was isolated using Trizol® Plus kit (Invitrogen) according to the manufacturer protocol.
Total RNA was treated with MICROBExpressTM (Ambion) to remove ribosomal RNA following the manufacturer protocol followed with the treatment 10 U of Tobacco Acid Pyrophosphatase (TAP) (epicentre) for 1.5 hours at 37°C. 10 pmol of RNA adapter (5'-ACACUCUUUCCCUACACGACGCUCUUCCGAUCU-3') was ligated using 25 U of T4 RNA Ligase 1 (New England BioLabs) for 12 h at 16°C. cDNA was then generated from purified RNA using SuperScript® II Reverse Transcriptase (Invitrogen) with 10 pmol of primer (5'-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNN-3') following standard manufacturer protocol and treated with 1 Unit of RNase H (Invitrogen) for 20 min at 37°C. cDNA was PCR amplified for 12 cycles using primers (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', 5'-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3'). DNA was size selected from 100bp to 300bp using agarose gel-electrophoresis, purified using QIAquick Gel Extraction Kit (Qiagen). 100ng of DNA was then treated with duplex-specific nuclease (evrogen) to remove ribosomal cDNA following standard manufacturer protocol and PCR amplified for 10 cycles.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
RACE |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Sequencing reads were mapped using Bowtie version 0.12.7 (Langmead et al., 2009) to the C. crescentus NA1000 reference genome (Genbank: CP001340). All valid alignments were made, and only reads that mapped to a unique location were used with no mismatches allowed. The position of each alignment is distributed into to the 5' most base for 5' end mapping
Aligned sequencing reads were normalization by the total number of non-ribosomal RNA reads for comparison.
The value of normalized sequencing reads obtained at each TSS position of each time point library was calculated.
Genome_build: NA1000: CP001340
Supplementary_files_format_and_content: Supplementary_files_format_and_content: Each line in the processed file time_point_TSS.txt has the following organization in columns: Col 1: Genomic coordinate of TSS, Col 2: chromosome strand of TSS, Col 3: t=0 normalized reads, Col 4: t=20 normalized reads, Col 5: t=40 normalized reads, Col 6: t=60 normalized reads, Col 7: t=80 normalized reads, Col 8: t=100 normalized reads, Col 9: t=120 normalized reads, Col 10: t=140 normalized reads
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| Submission date |
May 06, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jared Michael Schrader |
| E-mail(s) |
schrader@wayne.edu
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| Organization name |
Wayne State University
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| Department |
Biological Sciences
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| Lab |
Schrader Lab
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| Street address |
5047 Gullen Mall, Biological Sciences Building Room 2168
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| City |
Detroit |
| State/province |
MI |
| ZIP/Postal code |
48202 |
| Country |
USA |
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| Platform ID |
GPL18275 |
| Series (2) |
| GSE57364 |
The Global Landscape of Transcription Initiation During the Caulobacter Cell Cycle [timecourse] |
| GSE57366 |
The Global Landscape of Transcription Initiation During the Caulobacter Cell Cycle |
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| Relations |
| BioSample |
SAMN02754412 |
| SRA |
SRX534369 |
| 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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