|
| Status |
Public on Jul 08, 2019 |
| Title |
Hfq_RIPseq: WTIN-1 |
| Sample type |
SRA |
| |
|
| Source name |
cells
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
genotype: rph-1
molecule subtype: Hfq IP input, fragmented RNA (rRNA-depleted)
|
| Growth protocol |
Strains were grown with aeration in LB broth at 37°C to an OD600 of 1.0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using hot phenol-chloroform and alcohol precipitation
Libraries of total RNA samples were generated by removing DNA by DNase treatment (DNase Turbo; Ambion), depleting rRNA using the Ribo-Zero rRNA Removal Kit for gram-negative bacteria (Illumina), and subsequently utilizing the TruSeq Stranded Total RNA Library Kit (Illumina). Libraries of Hfq input and IP samples were generated by applying the RNA Fragmentation Kit (Ambion), removing 5' triphosphates via RNA 5' polyphosphatase (Epicenter), and subsequently utilizing the TruSeq Small RNA Library Kit (Illumina).
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
processed data file : none
|
| Data processing |
Illumina adapters and low-quality ends < 20 Phred were trimmed using Cutadapt version version 1.16 with the parameters -m 18 -q 20,20 -a AGATCGGAAGAGC -A AGATCGGAAGAGC. Reads were mapped to the Escherichia coli MG1655 genome (NC_000913.3) using bwa-mem version 0.7.13-r1126 with the parameters -t 24 -v 3 -r 1 -I 150,50,600,1.
Quantification of gene counts was performed using featureCounts version 1.6.2 using parameters -T 24 -s 2 -p -B -C. Differential gene expression analysis and normalized gene counts were calculated using the DESeq2 R package, version 1.22.1.
Identification of mRNA fragments in Hfq input samples was performed by using DESeq2 to identify significantly increased expression in the Δpnp Hfq Input versus the wild type Hfq Input over 5 nt segments tiled over the entire genome. Contiguous segments of 60 nt or greater were merged as individual fragments, and those fragments with a log2 fold change > 1.5, a z-score of fragment versus local Δpnp coverage > 1.0, and average Δpnp coverage > 300 were retained.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: Supplemental Table S3.xlsx: differentially expressed genes. Supplemental Table S4.xlsx: mRNA fragments upregulated in the absence of pnp. counts_HfqIP_WTIN_PNIN_WTIP_PNIP.txt: gene counts for WTIN, PNIN, WTIP, and PNIP. counts_KR10000_NRD999.txt: gene counts for KR10000 and NRD999.
|
| |
|
| Submission date |
Jan 18, 2019 |
| Last update date |
Jul 09, 2019 |
| Contact name |
Nicholas De Lay |
| E-mail(s) |
nicholas.r.delay@uth.tmc.edu
|
| Phone |
713-500-6293
|
| Organization name |
University of Texas Health Science Center
|
| Department |
Microbiology and Molecular Genetics
|
| Street address |
6431 Fannin St
|
| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL15010 |
| Series (1) |
| GSE125368 |
Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments |
|
| Relations |
| BioSample |
SAMN10777048 |
| SRA |
SRX5263781 |
