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Sample GSM1378471

Query DataSets for GSM1378471

Status

Public on Aug 04, 2014

Title

CK-2

Sample type

SRA

Source name

liquid culture in LB

Organism

Pseudomonas aeruginosa

Characteristics

strain: MPAO1/pAK1900
chip antibody: anti-VSV

Treatment protocol

grow until mid-log phase
grow until mid-log phase

Growth protocol

LB medium with Amp

Extracted molecule

genomic DNA

Extraction protocol

Wild type P. aeruginosa MAPO1 containing empty pAK1900 or pAK1900-VqsM-VSV was cultured in LB medium supplemented with ampicillin until the mid-log phase (OD=0.6), before was treated with 1% formaldehyde for 10 min at 37 °C. Cross-linking was stopped by addition of 125 mM glycine. Bacterial pellets were washed twice with a Tris buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl), and then re-suspended in 500 µl 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% SDS, mini-protease inhibitor cocktail (Roche) and sonicated the DNA to sizes of 100-300 bp. Insoluble cellular debris was removed by centrifugation and the supernatant used as input sample in IP experiments. Both control and IP samples were washed by protein A beads (General Electric), and then incubated with 50 ul agarose-conjugated anti-VSV antibodies (Sigma) in IP buffer. Washing, crosslink reversal, and purification of the ChIP DNA were conducted by following previously published protocols.
NEXTflex™ ChIP-Seq Kit (Bioo Scientific)
Wild type P. aeruginosa MAPO1 containing empty pAK1900 or pAK1900-VqsM-VSV was cultured in LB medium supplemented with ampicillin until the mid-log phase (OD=0.6), before was treated with 1% formaldehyde for 10 min at 37 °C. Cross-linking was stopped by addition of 125 mM glycine. Bacterial pellets were washed twice with a Tris buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl), and then re-suspended in 500 µl 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% SDS, mini-protease inhibitor cocktail (Roche) and sonicated the DNA to sizes of 100-300 bp. Insoluble cellular debris was removed by centrifugation and the supernatant used as input sample in IP experiments. Both control and IP samples were washed by protein A beads (General Electric), and then incubated with 50 ul agarose-conjugated anti-VSV antibodies (Sigma) in IP buffer. Washing, crosslink reversal, and purification of the ChIP DNA were conducted by following previously published protocols.
NEXTflex™ ChIP-Seq Kit (Bioo Scientific)

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Bacterial ChIP-seq reads were mapped to the P. auruginosa genomes, using TopHat (version 2.0.0) with two mismatches allowed.
Only the uniquely mapped reads were kept for the subsequent analyses.
The enriched peaks were identified using MACS software (version 2.0.0)
Genome_build: P. aeruginosa PAO1

Submission date

May 05, 2014

Last update date

May 15, 2019

Contact name

Xin Deng

E-mail(s)

xindeng@cityu.edu.hk

Phone

852-34425693

Organization name

City University of Hong Kong