|
| Status |
Public on Nov 13, 2023 |
| Title |
dely_ip_an_1 |
| Sample type |
SRA |
| |
|
| Source name |
hqbA (XL678)
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: hqbA (XL678)
growth condition: anaerobically
treatment: Hfq co-IP RNA
|
| Treatment protocol |
The pellet of one aliquot was resuspended in the same volume of medium and grew continuously under aerobic condition. The other aliquot was moved to an anaerobic chamber and resuspended in the same volume of deoxygenated LB with NaNO3 (5 mM) and grew anaerobically.
|
| Growth protocol |
The strains were cultured in LB supplemented with 5 mM NaNO3 at 37 °C with agitation. When the OD600 reached 0.3, each culture was split into two aliquots and pelleted by centrifuging at 4500 rpm for 10 min at room temperature.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
When the OD600 reached 0.9 to 1.0, 20 OD600 of cells were collected and lysed by vortexing with 212-300 mm glass beads (Sigma) in a total volume of 2 ml lysis buffer (20 mM Tris-HCl/pH 8.0, 150 mM KCl, 1 mM MgCl2 and 1 mM DTT). The isolation of total RNA and Hfq-IP RNA was performed as previously described 38 with following modifications. 1.9 ml of cell lysate were mixed with 200 ml of Hfq antibody and 240 mg of protein A-Sepharose CL-4B (GE Healthcare). Hfq-IP RNA was isolated from protein A-Sepharose beads by extraction with phenol : chloroform : isoamyl alcohol (25:24:1), followed by ethanol precipitation. Total RNA was isolated from 100 ml of cell lysate by Trizol (ThermoFisher Sciientific) extraction followed by chloroform extraction and isopropanol precipitation. RNA were dissolved in 15 ml of DEPC water.
cDNA libraries were constructed based on a protocol adapted to capture the bacterial sRNAs 57. 400 ng RNA samples were used as input and the index primers were listed in Table S3. 7 cycles of PCR were performed at the last step to enrich each library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
The Illumina reads in FASTQ format were first trimmed by Cutadapt 58 to remove the adaptor sequences and low quality reads.
After quality control with FASTQC 0.11.9, the trimmed reads were processed with the RNA-Seq pipeline READemption version 2.0.3.
Reads longer than 12 nucleotides were aligned to the Escherichia coli str. K-12 substr. MG1655 reference sequence (NC_000913.3) from the NCBI. In addition to the known genes and small RNAs in the annotation published previously, we manually curated 87 tRNA genes and operon precursors by comparing the sequences and the annotations in EcoCyc (https://ecocyc.org), further discussed in supplemental materials and methods.
Nucleotide-wise coverage files in wiggle format and gene-wise normalized read counts were generated for visualization and comparison. Differential gene expression analysis was performed with DESeq2 using fold-change of 2.0 or higher and the adjusted p-value below 0.1 as the threshold.
Assembly: Ecoli K-12 MG1655 NC_000913.3
Supplementary files format and content: YhbS-HqbA.RawCount.RPKM.xlsx which contains 3 sheets of the raw count and RPKM, corresponding to the S4, S5 and S7 tables of the manuscropt aerobic, anaerobic and tRNA only data.
Supplementary files format and content: E.coli_K12_MG1655_NC000913.3_tRNA_annotation.xlsx is the E.coli K12 MG1655 tRNA annotation manually curated by Dr. Susan Gottesman.
|
| |
|
| Submission date |
Jun 16, 2023 |
| Last update date |
Nov 13, 2023 |
| Contact name |
Susan Gottesman |
| Organization name |
NCI/NIH
|
| Street address |
37 Convent Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL18956 |
| Series (1) |
| GSE235194 |
A novel acetyltransferase regulates the RNA binding capacity of the RNA chaperone Hfq in Escherichia coli |
|
| Relations |
| BioSample |
SAMN35778387 |
| SRA |
SRX20710901 |
