|
| Status |
Public on Oct 15, 2014 |
| Title |
tap+ |
| Sample type |
SRA |
| |
|
| Source name |
Total RNA lystate
|
| Organism |
Caulobacter vibrioides NA1000 |
| Characteristics |
strain: NA1000
|
| 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 doi:10.1016/0076-6879(91)04019-K)
|
| 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 in tap+. To tap+ and tap- samples, 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
tap+ and tap- aligned sequencing reads were normalization by the total number of non-ribosomal RNA reads for comparison.
The log value of the ratio (θ) between the number of sequencing reads obtained at the 5’ ends of tap+ and tap- libraries was calculated for each mapped genomic location
Genomic locations that meet the following criteria are identified as transcriptional start sites (TSS): (1) normalized tap+ read > 25 (2) θ > 0.26 (3) (3) An increase of >35% of RNA-seq density (Schrader et al, 2014) in the region 0-38bp upstream TSS than 1-38bp in the region downstream of TSS. (4) have been biochemical validated
Genome_build: NA1000: CP001340
Supplementary_files_format_and_content: Supplementary_files_format_and_content: Each line in the processed file tap_TSS.txt has three columns: Col 1: Genomic coordinate of TSS identifed based on CP001340, Col 2: chromosome strand of TSS, Col 3: normalized tap+ reads, Col 4: normalized tap- reads
|
| |
|
| Submission date |
May 06, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jared Michael Schrader |
| E-mail(s) |
schrader@wayne.edu
|
| 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) |
| GSE57365 |
The Global Landscape of Transcription Initiation During the Caulobacter Cell Cycle [mapping] |
| GSE57366 |
The Global Landscape of Transcription Initiation During the Caulobacter Cell Cycle |
|
| Relations |
| BioSample |
SAMN02754414 |
| SRA |
SRX534372 |
