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Sample GSM613804 Query DataSets for GSM613804
Status Public on Feb 05, 2011
Title [E-MTAB-332] H-NS early-exponential
Sample type SRA
 
Source name H-NS, EE
Organism Escherichia coli
Characteristics material type: whole_organism
strainorline: K-12 substr. MG1655
genotype: hns::3xFLAG
growth condition: early-exponential
chip antibody: FLAG antibody
Growth protocol grow | The E. coli K-12 MG1655 bacterial strains used in this work are the following: E. coli MG1655 (F- lambda- ilvG- rfb-50 rph-1); MG1655 hns (hns::Kanr); MG1655 fis (fis::Kanr); MG1655 hns-FLAG (hns::3xFLAG::Kanr); MG1655 fis-FLAG (fis::3xFLAG::Kanr). Luria-Bertani (0.5% NaCl) broth and agar (15 g/liter) were used for routine growth. Where needed, ampicillin, kanamycin, and chloramphenicol were used at final concentrations of 100, 30, and 30 ug/ml respectively.
Extracted molecule genomic DNA
Extraction protocol nucleic_acid_extraction | ChIP was performed as previously described (Grainger et al, 2004) with some modifications to the protdegree Col. Cells were grown aerobically at 37degree C to the desired OD600 and formaldehyde was added to a final concentration of 1%. After 20 min of incubation, glycine was added to a final concentration of 0.5 M to quench the reaction and incubated for a further 5 min. Cross-linked cells were harvested by centrifugation and washed twice with ice-cold TBS (pH 7.5). Cells were resuspended in 1 ml of 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 37degree C for 30 min. Following 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 sulfate [SDS] and PMSF [final concentration 1 mM]) was added and the lysate passed through a French pressure cell twice. 2 ml aliquots were removed and the DNA sheared to an average size of ~200 bp using a Bioruptor (Diagenode) with 30 cycles of 30 sec on/off at high setting. Insoluble cellular matter was removed by centrifugation for 10 min at 4degree C, and the supernatant was split into two 800 microl aliquots. The remaining 400 microl was kept to check the size of the DNA fragments.<br><br>Each 800 microl aliquot was incubated with 20 microl Protein A/G UltraLink Resin (Pierce) on a rotary shaker for 45 min at room temperature to get rid of complexes binding to the resin non-specifically. The supernatant was then removed and incubated with either no antibody (mdegree Ck-IP), FLAG mouse mondegree Clonal antibody (Sigma-Aldrich) or RNAP Beta subunit mouse mondegree Clonal (Nedegree Clone) and 30 microl Protein A/G UltraLink Resin, pre-incubated with 1mg/ml bovine serum albumin (BSA) in TBS, on a rotary shaker at 4degree C overnight (FLAG antibody) or at room temperature for 90 min (RNAP Beta subunit antibody). 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 microl elution buffer (10 mM Tris [pH 7.5], 10 mM EDTA and 1% SDS) at 65degree C for 20 min.
sequencing| Immunoprecipitated samples and the sheared DNA following the Bioruptor were de-crosslinked in 0.5x elution buffer containing 0.8 mg/ml Pronase at 42degree C for 2 h followed by 65degree C for 6 h. DNA was purified using a PCR purification kit (QIAGEN). Prior to sequencing, the DNA fragment sizes were checked and gene-specific quantitative PCR (qPCR) was carried out. Prior and post library construction, the concentration of the immunoprecipitated DNA samples was measured using the Qubit HS DNA kit (Invitrogen). Library construction and sequencing was done using the ChIP-Seq Sample Prep kit, Reagent Preparation kit and Cluster Station kit (Illumina). Samples were loaded at a concentration of 10 pM.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer II
 
Description Provider: EMBL_Heidelberg
Data processing Sequences obtained from the Illumina Genome Analyzer were mapped to both strands of the E. coli K12 MG1655 genome using BLAT allowing no gaps and up to two mismatches. Each alignment was extended to 200bp the approximate average length of DNA fragments on the 3' end. Only reads which mapped to a single region of the genome were considered for further analysis. For each base position on the genome, the number of reads that mapped to that position was calculated.
 
Submission date Oct 28, 2010
Last update date May 15, 2019
Organization European Bioinformatics Institute
E-mail(s) miamexpress@ebi.ac.uk
Lab ArrayExpress
Street address Wellcome Trust Genome Campus
City Hinxton
State/province Cambridgeshire
ZIP/Postal code CB10 1SD
Country United Kingdom
 
Platform ID GPL10328
Series (1)
GSE24991 [E-MTAB-332] H-NS and Fis binding to E. coli K12
Relations
SRA ERX006211

Supplementary file Size Download File type/resource
GSM613804_HnsA.blat.basecount.pruned.txt.gz 14.7 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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