 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 08, 2014 |
| Title |
WT_16_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
WT_16_rep1
|
| Organism |
Escherichia coli K-12 |
| Characteristics |
sub strain: BW38028
phase of growth: Late log phase OD 1.6
rna treatment: no RNA treatment
strain: K-12
|
| Treatment protocol |
Prior to total RNA extraction harvested bacterial cells were stored at -80.0°C in an equal volume of RNAlater.
|
| Growth protocol |
Wild type E. coli K-12 (strain BW38038) and BW39452(ΔrpoS) cultures were grown on MOPS glucose minimal medium with 0.2% glucose as sole carbon source in a 2L B. Braun Biostat® B fermenter with working volume of 1 L MOPS minimal medium with 0.2% glucose, at 37°C, pH was kept constant at 7.4 by the addition of 1 M NaOH, and dissolved oxygen was maintained above 40% of saturation by adjusting the agitation speeds in the range of 270–500 rpm with fixed 1.5 liter/min air flow. Culture samples were harvested by using a homemade sampling device seven times during logarithmic growth and three times following entry into stationary phase for the WT and two times during logarithmic phase and three times during stationary phase for the rpoS mutant. OD600 measurements were made on a Beckman Coulter DU 800 spectrophotometer. Samples were harvested directly into ice-cold RNAlater at a 1:1 dilution to protect RNA from degradation and cells then were pelleted by centrifugation at 8000rpm for 10 minutes. Cell pellets were stored at -80°C in an equal volume of RNAlater prior to RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All samples were extracted using the Qiagen Rnasey Mini Kit, following standard protocol
cDNA libraries were constructed at Purdue using an adapted SOLiD Total RNA-Seq Kit. Total RNA (DNase I digested) was fragmented by RNase III. RNA samples labeled as TEX were also subsequently digested with Terminator Exonuclease (TEX). Pyrophosphate groups were removed from the 5′ terminus using tobacco acid pyrophosphatase (TAP), and an RNA adapter was ligated to the 5′ end of the RNA. First-strand synthesis was performed using standard SOLiD 4 Total RNA-Seq protocol. RNA-seq via SOLiD 4 sequencing of libraries prepared by ligation based chemistry to provide strand-specific datasets.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD 4 System |
| |
|
| Data processing |
The raw data output (XSEQ files) from the SOLiD 4 Genetic Analyzer System are passed through the ABI Sequence Accuracy Enhancement Tool (SAET)
For alignment of the SAET reads to the E. coli MG1655 reference genome (RefSeq NC_000913), the short read alignment tool Bowtie ver. 1.8 (Langmead, et al., PMID 19261174) was utilized in three consecutive passes for each sample dataset. For the first pass, we use paired end color space mapping with a distance cutoff of 350 bases between read mates. Bowtie parameters were set to include only perfect matches and suppress reads that map to more than one genome location, i.e., uniquely mapped reads are retained. In practice we found the efficiency of paired end mapping was between 3 and 10%. To improve the overall alignment we mapped the orphan 5’ and 3’ end reads in two additional passes with Bowtie. The output of the three passes through Bowtie was three SAM files for each sample. Overall, we achieved 40-60% mapping efficiency with this three-pass strategy.
Used SAMTOOLS (Li, et al., PMID 1950593) to sort and index the SAM files obtained from Bowtie and convert them to BAM format.
Sequence data was processed by conversion of the sample alignment (BAM) files to strand-specific base count (WIG) files. To accomplish this an in-house script was created to extract strand-specific base count data from BAM files (outputs are positive and negative strand WIG files). First, the script reads in the paired-end BAM file and counts the nucleotides spanning inserts between the mated 5’ and 3’ reads. Next, the script pulls in the orphan 5’ and 3’ reads from the respective BAM files and increments the base counts at each base location without duplicating the reads already incremented from the paired ends.
WIG files were normalized by using an in-house script that reads in the raw WIG files while excluding counts from all 22 rRNA genes. A simple global normalization approach was utilized that multiplied the count at each base location by 1 billion and divides that value by the sum of base counts at all base locations in the file. This normalization strategy is anlogous to the Total Count approach used for normalizing gene-specific read alignments. In this way, the base counts are expressed as parts per billion.
WIG files were viewed and annotated using Jbrowse and Integrated Genome Viewer.
Genome_build: ASM584v1; Reference genome for E. coli MG1655 (RefSeq NC_000913). Paper title:Escherichia coli K-12: a cooperatively developed annotation snapshot--2005
Supplementary_files_format_and_content: WIG files are provided showing uniquely mapped sequence reads (normalized).
|
| |
|
| Submission date |
Nov 04, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
James Creecy |
| E-mail(s) |
jcreecy@ou.edu
|
| Organization name |
University of Oklahoma
|
| Department |
Microbiology
|
| Lab |
T. Conway
|
| Street address |
770 Van Vleet Oval
|
| City |
Norman |
| State/province |
Oklahoma |
| ZIP/Postal code |
73019 |
| Country |
USA |
| |
|
| Platform ID |
GPL17884 |
| Series (1) |
| GSE52059 |
Unexpected complexity of the Escherichia coli K-12 transcriptome architecture revealed by single nucleotide resolution RNA sequencing |
|
| Relations |
| BioSample |
SAMN02391512 |
| SRA |
SRX373115 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1258057_WT_16_rep1_neg.wig.gz |
2.8 Mb |
(ftp)(http) |
WIG |
| GSM1258057_WT_16_rep1_pos.wig.gz |
2.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
