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Sample GSM1936195

Query DataSets for GSM1936195

Status

Public on Jun 10, 2016

Title

wt_2_IP

Sample type

SRA

Source name

Bacterial cells

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

genotype: MG1655 wild-type
chip antibody: RpoS (Neoclone cat. no. WP009)

Treatment protocol

Formaldehyde was added to a final concentration of 1%. After incubation for 20 minutes, glycine was added to a final concentration of 0.5 M and incubated for 5 minutes.

Growth protocol

Cells were grown at 37°C, 200 rpm in M9 glucose, for 16 hours (stationary phase).

Extracted molecule

genomic DNA

Extraction protocol

Cross-linked cells were harvested by centrifugation, washed thrice with ice-cold TBS (pH 7.5), resuspended in 1 ml lysis buffer [10 mM Tris (pH 8.0), 20% sucrose, 50 mM NaCl, 10 mM EDTA, 20 mg/ml lysozyme and 0.1 mg/ml RNase A] and incubated at 37°C for 30 min. After lysis, 3 ml immunoprecipitation (IP) buffer [50 mM HEPES–KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and PMSF (final 1 mM)] was added and the DNA sheared to an average size of ~250 bp using a Bioruptor (Diagenode). Insoluble cellular matter was removed by centrifugation for 10 min at 4°C. An 800 μl aliquot was incubated with 20 μl Protein A/G UltraLink Resin (Pierce) on a rotary shaker for 45 minutes at room temperature. The supernatant was removed and incubated with mouse monoclonal antibody (Neoclone cat. no. WP009) and 30 μl Protein A/G UltraLink Resin (pre-incubated with 1mg/ml BSA in TBS), on a rotary shaker at room temperature for 90 min. Samples were washed once with IP buffer, twice with IP buffer + 500 mM NaCl, once with wash buffer [10 mM Tris (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet P-40 and 0.5% sodium deoxycholate] and once with TE (pH 7.5). Immunoprecipitated complexes were eluted in 100 μl elution buffer [10 mM Tris (pH 7.5), 10 mM EDTA and 1% SDS] at 65°C for 20 min. Immunoprecipitated samples and the sheared DNA from the Bioruptor were uncrosslinked in elution buffer containing 0.8 mg/ml Pronase at 42°C for 2 h followed by 65°C for 6 h. DNA was purified using the phenol-chloroform method.
Libraries were prepared for sequencing using standard Illumina protocols

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

w2_peaks.narrowPeak

Data processing

Reads were trimmed for quality using Trimmomatic-0.32 with a cutoff quality score of 20
Raw sequence data obtained in the fastq format were aligned to the genome of E. coli K12 MG1655 (NC_000913.2) using the Burrows-Wheeler matching program BWA. Reads mapping uniquely to the genome were selected. Files were coverted to .bam format using samtools. The MACS 2.1.0 software was used to call peaks. Peaks overlapping in both replicates were selected for further analysis.
Genome_build: NC_000913.2
Supplementary_files_format_and_content: .narrowPeak files output by MACS, giving peak start and end positions, as well as fold-change, -log10pvalue, -log10qvalue, and relative summit position to peak start

Submission date

Nov 09, 2015

Last update date

May 15, 2019

Contact name

Avantika Lal

E-mail(s)

avantika@ncbs.res.in

Organization name

National Centre for Biological Sciences

Street address

GKVK, Bellary Road