<?xml version="1.0" encoding="UTF-8"?>

<gse xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:noNamespaceSchemaLocation="esquema-gcs.xsd">

^DATABASE = GeoMiame
!Database_name = Gene Expression Omnibus (GEO)
!Database_institute = NCBI NLM NIH
!Database_web_link = http://www.ncbi.nlm.nih.gov/geo
!Database_email = geo@ncbi.nlm.nih.gov
^SERIES = GSE88979
!Series_title = Revealing the genome-scale transcriptional regulatory landscape of OmpR highlights its expanded regulatory roles and unexpected importance of narU under osmotic stress in Escherichia coli K-12 MG1655 [ChIP-exo]
!Series_geo_accession = GSE88979
!Series_status = Public on Jun 09 2017
!Series_submission_date = Oct 20 2016
!Series_last_update_date = Jun 09 2017
!Series_pubmed_id = 28526842
!Series_summary = A transcription factor (TF), OmpR, plays a critical role in transcriptional regulation of the defense system for osmotic stress in bacteria. However, its full genome-wide regulatory potential is unknown. Here, we perform a genome-scale reconstruction of the OmpR regulon in Escherichia coli K-12 MG1655. Integrative data analysis reveals that a total of 37 genes in 24 transcription units (TUs) belong to OmpR regulon. Among them, 26 genes show more than two-fold changes in expression level under OmpR knock-out condition. We find that OmpR tends to regulate mostly membrane-located gene products of diverse fundamental biological processes, such as narU, ompX, and nuoN. Investigating co-regulation of entire set of genes regulated by other stress-response TFs unveils that they are surprisingly independently regulated by TF(s) responding to each stress. Additionally, detailed investigation of physiological roles of newly discovered OmpR regulon reveals that activation of narU encoding nitrate/nitrite transporter is a relatively unique strategy of E. coli K-12 MG1655 to significantly improve cellular tolerance toward osmotic stress.
!Series_overall_design = A total of two samples were analyzed. ompR-8myc tagged cells were cultured in M9 minimal media with 0.2% glucose. Then cells were treated with 0.3 M of NaCl at mid-log pahse for 30 min with agitation.
!Series_type = Genome binding/occupancy profiling by high throughput sequencing
!Series_contributor = Sang-Woo,,Seo
!Series_contributor = Donghyuk,,Kim
!Series_sample_id = GSM2356685
!Series_sample_id = GSM2356686
!Series_contact_name = Donghyuk,,Kim
!Series_contact_email = donghyuk.kim@khu.ac.kr
!Series_contact_laboratory = Systems Biology Lab
!Series_contact_department = Department of Genetic Engineering
!Series_contact_institute = Kyung Hee University
!Series_contact_address = 1732 Deokyoung-daero, Giheung-gu
!Series_contact_city = Yongin-si
!Series_contact_state = Gyeonggi-do
!Series_contact_zip/postal_code = 17104
!Series_contact_country = South Korea
!Series_supplementary_file = ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE88nnn/GSE88979/suppl/GSE88979_RAW.tar
!Series_platform_id = GPL17439
!Series_platform_taxid = 511145
!Series_sample_taxid = 511145
!Series_relation = SubSeries of: GSE88981
!Series_relation = BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA349459
!Series_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP091859
^PLATFORM = GPL17439
!Platform_title = Illumina MiSeq (Escherichia coli str. K-12 substr. MG1655)
!Platform_geo_accession = GPL17439
!Platform_status = Public on Jul 12 2013
!Platform_submission_date = Jul 12 2013
!Platform_last_update_date = Jul 12 2013
!Platform_technology = high-throughput sequencing
!Platform_distribution = virtual
!Platform_organism = Escherichia coli str. K-12 substr. MG1655
!Platform_taxid = 511145
!Platform_contact_name = ,,GEO
!Platform_contact_country = USA
!Platform_data_row_count = 0
^SAMPLE = GSM2356685
!Sample_title = <Name>OmpR NaCl 1</Name>
!Sample_geo_accession = GSM2356685
!Sample_status = Public on Jun 09 2017
!Sample_submission_date = Oct 20 2016
!Sample_last_update_date = Jun 09 2017
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = OmpR NaCl 1
!Sample_organism_ch1 = <Orgn>Escherichia coli</Orgn> str. <Strain>K-12</Strain> substr. <Substrain>MG1655</Substrain>
!Sample_taxid_ch1 = 511145
!Sample_characteristics_ch1 = strain: K-12 MG1655
!Sample_characteristics_ch1 = genotype: <Gtype>ompR-8myc</Gtype>
!Sample_characteristics_ch1 = chip antibody: <Anti>anti-myc</Anti> (Santa Cruz Biotech, sc-28207)
!Sample_treatment_protocol_ch1 = E. coli cells were crosslinked in 1% formaldehyde for 25 minutes at RT on a rocker, then washed 3 times with 50 ml of ice-cold TBS (Tris buffered saline) each time.
!Sample_growth_protocol_ch1 = A total of two samples were analyzed. ompR-8myc tagged cells were cultured in <Med>M9 minimal media</Med> with <Supp>0.2% glucose</Supp>. Then cells were treated with <Supp>0.3 M of NaCl</Supp> at <Phase>mid-log pahse</Phase> for <Supp>30 min</Supp> with agitation.
!Sample_molecule_ch1 = genomic DNA
!Sample_extract_protocol_ch1 = Crosslinked cells were then resuspended in 500 ul of lysis buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA) with 40 ul of protease inhibitor cocktail (50 mg in 0.25 ml of DMSO and 0.75 ml of TDW). Cells were lyzed with 1 ul of lysozyme for 30 min at 37oC on a rocker. 0.55 ml of 2X IP buffer (100 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2% Triton X-100 and 1 mM EDTA) were added to the sample, and then was sonicated to fragmentize genomic DNA. 0.3 ml of Wash buffer I (50 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1% Triton X-100 and 1mM EDTA) was added to make the volume up to 1.4 ml. Only 0.7 ml was taken and transfered to a new tube, and 15 ul of Anti-c-myc mouse antibody was added, and the sample was incubated overnight at 4 oC to make Antibody-TF complex. 50 ul of Dynabeads Pan mouse IgG were washed 3 times with bead washing solution (250 mg BSA in 50 ml of PBS), and were added to the sample. Cell lysate with beads were incubated for 6 hours or overnight at 4oC to make Dynabead-antibody-TF complex. The beads were pulled down on a magnet stand, and washed 2 times with wash buffer I and with wash buffer II (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1% Triton X-100 and 1mM EDTA), wash buffer III (10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1% Triton X-100 and 1mM EDTA), and wash buffer IV (10 mM Tris-HCl (pH 8.0), 1mM EDTA). The bead-bound TF-DNA complex was then end-repaired, dA-tailed, and ligated to the first adapter. Adapter-ligated sample was then treated with nick-repair reagent, and was treated with lambda exonuclease and RecJ exonuclease. Then DNA was eluted away from Dynabeads by incubating in 200 ul of elution buffer (50 mM Tris-HCl (pH 8.0), 1% SDS and 1 mM EDTA) at 65oC overnight. Protein was removed by treating 4 ul of protease K and being incubated at 55 oC for 2 hours, and by Phenol-Chloroform-IAA extraction. Purified DNA was used to bulid the second strand synthesis, followed by another dA-tailing, second strand ligation, and 3' overhang removal stpes. Then the sequencing library was amplified with PCR enrichment.
!Sample_extract_protocol_ch1 = ChIP-seq libraries were prepared for sequencing using standard Illumina protocols
!Sample_description = Immunoprecipitated DNA
!Sample_data_processing = ChIP-exo reads were aligned to the ASM584v2 genome reference sequence using using bowtie v1.0.0 with parameters  -S
!Sample_data_processing = MACE software (https://code.google.com/p/chip-exo/) was used to detect peaks with aligned output from bowtie mapping.
!Sample_data_processing = Read count was calculated for each genomic position from sequence alignment, and 95% strongest intensity was used as background intensity.
!Sample_data_processing = For each peak detected with MACE, binding intensity was calculated by averaging read counts from two biological replicates and dividing by background intensity.
!Sample_data_processing = Genome_build: <Gversion>ASM584v2</Gversion>
!Sample_data_processing = Supplementary_files_format_and_content: Tab-delimited text files in gff format which has 8 columns: sequence id, source(empty), feature (+/- strand), start position, end position, intensity score, strand(+/-), frame(.), attribute(.).
!Sample_platform_id = GPL17439
!Sample_contact_name = Donghyuk,,Kim
!Sample_contact_email = donghyuk.kim@khu.ac.kr
!Sample_contact_laboratory = Systems Biology Lab
!Sample_contact_department = Department of Genetic Engineering
!Sample_contact_institute = Kyung Hee University
!Sample_contact_address = 1732 Deokyoung-daero, Giheung-gu
!Sample_contact_city = Yongin-si
!Sample_contact_state = Gyeonggi-do
!Sample_contact_zip/postal_code = 17104
!Sample_contact_country = South Korea
!Sample_instrument_model = Illumina MiSeq
!Sample_library_selection = ChIP
!Sample_library_source = genomic
!Sample_library_strategy = <Technique>ChIP-Seq</Technique>
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN05930262
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX2254710
!Sample_supplementary_file_1 = ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM2356nnn/GSM2356685/suppl/GSM2356685_ompr_nacl1.gff.gz
!Sample_series_id = GSE88979
!Sample_series_id = GSE88981
!Sample_data_row_count = 0
^SAMPLE = GSM2356686
!Sample_title = <Name>OmpR NaCl 2</Name>
!Sample_geo_accession = GSM2356686
!Sample_status = Public on Jun 09 2017
!Sample_submission_date = Oct 20 2016
!Sample_last_update_date = Jun 09 2017
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = OmpR NaCl 2
!Sample_organism_ch1 = <Orgn>Escherichia coli</Orgn> str. <Strain>K-12</Strain> substr. <Substrain>MG1655</Substrain>
!Sample_taxid_ch1 = 511145
!Sample_characteristics_ch1 = strain: K-12 MG1655
!Sample_characteristics_ch1 = genotype: <Gtype>ompR-8myc</Gtype>
!Sample_characteristics_ch1 = chip antibody: <Anti>anti-myc</Anti> (Santa Cruz Biotech, sc-28207)
!Sample_treatment_protocol_ch1 = E. coli cells were crosslinked in 1% formaldehyde for 25 minutes at RT on a rocker, then washed 3 times with 50 ml of ice-cold TBS (Tris buffered saline) each time.
!Sample_growth_protocol_ch1 = A total of two samples were analyzed. ompR-8myc tagged cells were cultured in <Med>M9 minimal media</Med> with <Supp>0.2% glucose</Supp>. Then cells were treated with <Supp>0.3 M of NaCl</Supp> at <Phase>mid-log pahse</Phase> for <Supp>30 min</Supp> with agitation.
!Sample_molecule_ch1 = genomic DNA
!Sample_extract_protocol_ch1 = Crosslinked cells were then resuspended in 500 ul of lysis buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA) with 40 ul of protease inhibitor cocktail (50 mg in 0.25 ml of DMSO and 0.75 ml of TDW). Cells were lyzed with 1 ul of lysozyme for 30 min at 37oC on a rocker. 0.55 ml of 2X IP buffer (100 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2% Triton X-100 and 1 mM EDTA) were added to the sample, and then was sonicated to fragmentize genomic DNA. 0.3 ml of Wash buffer I (50 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1% Triton X-100 and 1mM EDTA) was added to make the volume up to 1.4 ml. Only 0.7 ml was taken and transfered to a new tube, and 15 ul of Anti-c-myc mouse antibody was added, and the sample was incubated overnight at 4 oC to make Antibody-TF complex. 50 ul of Dynabeads Pan mouse IgG were washed 3 times with bead washing solution (250 mg BSA in 50 ml of PBS), and were added to the sample. Cell lysate with beads were incubated for 6 hours or overnight at 4oC to make Dynabead-antibody-TF complex. The beads were pulled down on a magnet stand, and washed 2 times with wash buffer I and with wash buffer II (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1% Triton X-100 and 1mM EDTA), wash buffer III (10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1% Triton X-100 and 1mM EDTA), and wash buffer IV (10 mM Tris-HCl (pH 8.0), 1mM EDTA). The bead-bound TF-DNA complex was then end-repaired, dA-tailed, and ligated to the first adapter. Adapter-ligated sample was then treated with nick-repair reagent, and was treated with lambda exonuclease and RecJ exonuclease. Then DNA was eluted away from Dynabeads by incubating in 200 ul of elution buffer (50 mM Tris-HCl (pH 8.0), 1% SDS and 1 mM EDTA) at 65oC overnight. Protein was removed by treating 4 ul of protease K and being incubated at 55 oC for 2 hours, and by Phenol-Chloroform-IAA extraction. Purified DNA was used to bulid the second strand synthesis, followed by another dA-tailing, second strand ligation, and 3' overhang removal stpes. Then the sequencing library was amplified with PCR enrichment.
!Sample_extract_protocol_ch1 = ChIP-seq libraries were prepared for sequencing using standard Illumina protocols
!Sample_description = Immunoprecipitated DNA
!Sample_data_processing = ChIP-exo reads were aligned to the ASM584v2 genome reference sequence using using bowtie v1.0.0 with parameters  -S
!Sample_data_processing = MACE software (https://code.google.com/p/chip-exo/) was used to detect peaks with aligned output from bowtie mapping.
!Sample_data_processing = Read count was calculated for each genomic position from sequence alignment, and 95% strongest intensity was used as background intensity.
!Sample_data_processing = For each peak detected with MACE, binding intensity was calculated by averaging read counts from two biological replicates and dividing by background intensity.
!Sample_data_processing = Genome_build: <Gversion>ASM584v2</Gversion>
!Sample_data_processing = Supplementary_files_format_and_content: Tab-delimited text files in gff format which has 8 columns: sequence id, source(empty), feature (+/- strand), start position, end position, intensity score, strand(+/-), frame(.), attribute(.).
!Sample_platform_id = GPL17439
!Sample_contact_name = Donghyuk,,Kim
!Sample_contact_email = donghyuk.kim@khu.ac.kr
!Sample_contact_laboratory = Systems Biology Lab
!Sample_contact_department = Department of Genetic Engineering
!Sample_contact_institute = Kyung Hee University
!Sample_contact_address = 1732 Deokyoung-daero, Giheung-gu
!Sample_contact_city = Yongin-si
!Sample_contact_state = Gyeonggi-do
!Sample_contact_zip/postal_code = 17104
!Sample_contact_country = South Korea
!Sample_instrument_model = Illumina MiSeq
!Sample_library_selection = ChIP
!Sample_library_source = genomic
!Sample_library_strategy = <Technique>ChIP-Seq</Technique>
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN05930261
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX2254711
!Sample_supplementary_file_1 = ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM2356nnn/GSM2356686/suppl/GSM2356686_ompr_nacl2.gff.gz
!Sample_series_id = GSE88979
!Sample_series_id = GSE88981
!Sample_data_row_count = 0

</gse>