|
| Status |
Public on May 01, 2020 |
| Title |
Second Replicate RNA-seq sample OD600=4.0 |
| Sample type |
SRA |
| |
|
| Source name |
Escherichia coli grown in LB
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
genotype: wildtype
optical density: OD 4.0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Guanidium thiocyanate phenol method. RNA integrity was assessed with the Prokaryote Total RNA Nano assay on a 2100 Bioanalyzer (Agilent). Genomic DNA was removed by incubating 10 μg of total RNA with 2U Turbo DNase (Ambion) in a 50 μl final volume for 30 minutes at 37°C in the presence of 10 U SuperaseIn RNase Inhibitor (Ambion). RNA was subsequently phenol-chloroform extracted and purified by ethanol-precipitation.
The RNA-seq libraries were generated using the Illumina TrueSeq protocol according to manufacturer's procedures.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
RNA-seq sample
Supplementary Table 1.xlsx
|
| Data processing |
Raw sequencing reads in fastq files were processed using a pipeline developed by Sander Granneman, which uses tools from the pyCRAC package (Webb et al., 2014a). pyCRAC versions. 1.3.2 to 1.4.4 were used for the analyes. The entire pipeline is available at https://bitbucket.org/sgrann/). The CRAC_pipeline_PE.py pipeline first demultiplexes the data using pyBarcodeFilter.py and the in-read barcode sequences found in the L5 5’ adapters. Flexbar then trims the reads to remove 3’-adapter sequences and poor quality nucleotides (Phred score <23). Using the random nucleotide information present in the L5 5’-adaptor sequences, the reads were collapsed to remove potential PCR duplicates. The reads were then mapped to the Escherichia coli MG1655 genome with Novoalign (www.novocraft.com). To determine which genes the reads overlapped with we generated an annotation file in the Gene Transfer Format (GTF). This file contains the start and end positions of each gene on the chromosome as well as what genomic features (i.e. sRNA, protein- coding, tRNA) it belongs to. To generate this file, we used the Rockhopper software (Tjaden, 2015) on E. coli rRNA-depleted total RNA-seq data (generated by Christel Sirocchi), a minimal GTF file obtained from ENSEMBL (without UTR information). The resulting GTF file contained information not only on the coding sequences, but also complete 5’ and 3’ UTR coordinates. PyReadCounters then used the novoalign output file and the GTF file to count the total number of unique cDNAs that mapped to each gene. RNA-seq data was generated by Novogene using the Illumina TruSeq protocol. The data were processed using the CRAC_pipeline_PE.py. For all the CLASH and RNA-seq data, we normalized the counts to transcripts per million (TPM). Processed and raw data files for these counts are provided.
Genome_build: Escherichia coli MG1655
Supplementary_files_format_and_content: TPM
|
| |
|
| Submission date |
Apr 29, 2019 |
| Last update date |
May 01, 2020 |
| Contact name |
Sander Granneman |
| E-mail(s) |
Sander.Granneman@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Centre for Synthetic and Systems Biology
|
| Lab |
Granneman lab
|
| Street address |
Mayfield Road, Kings Buildings, Waddington building, room 3.06
|
| City |
Edinburgh |
| ZIP/Postal code |
EH9 3JD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL26592 |
| Series (2) |
| GSE123048 |
Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [RNA-seq] |
| GSE123050 |
Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation |
|
| Relations |
| BioSample |
SAMN11528403 |
| SRA |
SRX5766153 |
