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Sample GSM4151179

Query DataSets for GSM4151179

Status

Public on Sep 17, 2021

Title

Hfq65_IP_Rep2

Sample type

SRA

Source name

E.coli K12 MG1655

Organism

Escherichia coli K-12

Characteristics

strain: MG1655
genotype: Hfq65 mutant
anti-hfq antibody: co-IP with anti-Hfq polyclonal antibodies

Growth protocol

All strains used in this study were grown at 37 oC under aerobic conditions in LB. Wherever required, the liquid and solid media was supplemented with Ampicillin (100 mg/ml). The bacteria used for the RNA Seq experiment were grown to OD600 =1 in LB.

Extracted molecule

total RNA

Extraction protocol

Polyclonal antibodies to Hfq were generated previously by immunizing rabbits with purified Hfq protein (Covance). RNAs that co-IP with Hfq were isolated as described previously(Zhang, A., et al., Mol Cell, 9, 11-22) with the following modifications. Overnight cultures of strains KK2440 (WT) and KK2448 (Hfq65) cells were grown to OD600 ~ 1.0 in LB medium. Cells corresponding to the equivalent of 40 OD600 were collected, and 2 ml cell lysates were prepared by vortexing with 212-300 μm glass beads (Sigma) in a final volume of 2 ml lysis buffer (20 mM Tris-HCl/pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT). Co-IPs were carried out either using 200 μl of α-Hfq, 240 mg of protein A-Sepharose CL-4B (GE Healthcare), and 1.9 ml of cell lysate. Co-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 μl of cell lysate by Trizol (Thermo Fisher Scientific) extraction followed by chloroform extraction and isopropanol precipitation. Total and co-IP RNA samples were resuspended in 30 μl and 50 μl of DEPC H2O. The integrity of RNA was checked on 1% agarose gel. The RNA were treated with DNAase I(100 μl) followed by phenol: Chloroform: IAA based purification and ethanol precipitation. The gDNA contamination was checked by PCR amplification. The DNA free RNA samples were then subjected to rRNA depletion using Ribo-zero kit and purified by using (2.5X)RNAClean XP Beads and isopropanol (1.5X). isopropanol ratio(1.5X) is critical to keeping short RNAs (>70 nt) om the RMA [pp;. The isopropanol and RNAClean XP bead ratios were both optimized in orger to remove nonligated adaptors while saving short RNA fragments.
The RNAs (400 ng) were used for cDNA library preparation. Library construction was carried out based on the RNAtag-Seq methodology (Shishkin, et al. 2015, Nat Methods, 12, 323-325), which was adapted to capture (optimized amount of isopropanol and RNAClean XP for short sRNA) bacterial sRNA (Melamed, S et al., 2018, Nat Protoc, 13, 1-33). To enrich the libraries, 12 cycles of PCR were carried out at the end step of enrichment. Bead purification was performed twice at the last step to ensure high purity of the library from the adaptor dimer.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

RNA co-IP with Hfq

Data processing

Illumina Hiseq 2500 System was used for sequencing.
The Illumina reads in FASTQ format were first trimmed by Trimmomtaic version 0.36 to remove the adaptor sequences and low quality reads.
The RNAseq pipeline READemption version 0.4.5. was then used to process those trimmed reads. Those longer than 12 nt were aligned to the Escherichia coli str. K-12 substr. MG1665 reference sequence (NC_000913.3) download from the NCBI ftp website by using segemehl version 0.2.0.
To find differentially expressed genes, the “deseq” subcommand in READemption executing DESeq2 version 1.20.0 was used with the fold-change of 2.0 or higher and the adjusted p-value below 0.1.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: tab-delimited text files include rawcount for each Sample.

Submission date

Nov 05, 2019

Last update date

Sep 17, 2021

Contact name

Kumaran Ramamurthi

Organization name

National Cancer Institute

Lab

Lab of Molecular Biology

Street address

37 Convent Drive