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Sample GSM1381197 Query DataSets for GSM1381197
Status Public on Oct 15, 2014
Title t=120
Sample type SRA
Source name Total RNA lystate
Organism Caulobacter vibrioides NA1000
Characteristics strain: NA1000
timepoint (minutes): 120
Treatment protocol Cells were pelleted and immediately frozen in liquid nitrogen
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.
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection RACE
Instrument model Illumina Genome Analyzer
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
Submission date May 06, 2014
Last update date May 15, 2019
Contact name Jared Michael Schrader
Organization name Wayne State University
Department Biological Sciences
Lab Schrader Lab
Street address 5047 Gullen Mall, Biological Sciences Building Room 2168
City Detroit
State/province MI
ZIP/Postal code 48202
Country USA
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
BioSample SAMN02754407
SRA SRX534370

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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