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| Status |
Public on Jan 09, 2018 |
| Title |
CF104.3.3_y2 |
| Sample type |
SRA |
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| Source name |
E. coli strain isolated from CF patient stool
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| Organism |
Escherichia coli |
| Characteristics |
individual: CF patient
disease state: cystic fibrosis
carbon source: Glycerol
replicate: 1
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| Growth protocol |
Experiments were performed at 37°C and followed three steps: seed culture, pre-culture and experimental culture. In the seed culture the cells were grown overnight in Luria Broth, then diluted 1:100 in either M9 minimal media with glycerol or glucose for the pre-cultures. Finally, the experimental cultures were started from the overnight grown pre-cultures containing the same carbon source at a normalized optical density at 600 nm (OD600) of 0.05. Cultures were harvested at mid-exponential phase, cells were immediately spun down and cell pellets stored at -80°C until processed.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol and chloroform in conjunction with Qiagen’s RNeasy Mini kit (Qiagen, Valencia, CA) following the A. Untergasser protocol (http://www.molbi.de/protocols/rna_prep_comb_trizol_v1_0.htm) with one modification: Phase Lock Gel tubes (Eppendorf, Westbury, NY) were used to better separate organic and aqueous phases after the addition of chloroform. DNA was digested with Ambion rDNAse I (Life Technologies, Carlsbad, CA) for 30 min at 37°C. Total RNA was further cleaned using the RNeasy Mini kit and quality was assessed by the RNA ScreenTape Assay on the TapeStation 2200 (Agilent Technologies, Santa Clara, CA). Samples with RIN values greater than 8.0 were treated with the Ambion MICROBExpress Bacterial mRNA Enrichment Kit (Life Technologies, Carlsbad, CA) and were used for library construction.
cDNA libraries were barcoded with the TruSeq RNA Sample Prep Kit v2 (Illumina Inc., San Diego, CA). Libraries were sequenced on the Illumina MiSeq to produce 50 bp single reads. All steps in library construction and sequencing were performed according to manufacturer’s standards.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Basecalling performed by MiSeq (RTA)
Map to MG1655 reference using BWA 0.7.4 (bwa mem, default parameters)
Filter for map quality >= 20 using Samtools 0.1.18
Filter out rRNA reads, multi-mapped reads, > 3 mismatches, and gapped mappings using custom Python script (FilterBAM2BAM from http://uwgenomics.org/downloads/RNA-Seq-1.1.tar.gz)
Compile annotated counts using custom Python script (ExtractMapCounts from http://uwgenomics.org/downloads/RNA-Seq-1.1.tar.gz)
Genome_build: Escherichia coli str. K-12 substr. MG1655 (U00096.3)
Supplementary_files_format_and_content: expression level files are tab-delimited text with columns as follows: index, hits, RPKM, RPM, start_position, end_position, strand, feature_type, reference_replicon (type), locus_tag, gene_name, product
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| Submission date |
Jan 08, 2018 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Samuel Miller |
| Organization name |
University of Washington
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| Street address |
1959 Pacific Ave Box 357710
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL16085 |
| Series (1) |
| GSE108846 |
Adaptation of commensal proliferating Escherichia coli to the intestinal tract of young children with cystic fibrosis |
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| Relations |
| BioSample |
SAMN08324738 |
| SRA |
SRX3540074 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2914313_MS2034059-50V2_1_1_GCCAAT_v_U00096.sorted.gtQ19.noRNA.sMapped.LT4ed.ungapped.map_counts.tab.gz |
241.4 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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