<?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 = GSE60546
!Series_title = In vivo probing of the DNA-binding Architecture by bacterial arginine repressor
!Series_geo_accession = GSE60546
!Series_status = Public on Apr 01 2015
!Series_submission_date = Aug 20 2014
!Series_last_update_date = Apr 01 2015
!Series_pubmed_id = 25735747
!Series_summary = Although DNA motifs recognized by the transcription factors (TFs) have been determined, challenges remain in probing in vivo architecture of TF-DNA complexes on a genome-wide scale. Here, we show in vivo architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using chromatin immunoprecipitation coupled with sequencing (ChIP-exo). The identified 62 ArgR-binding loci were classified into three groups, comprised of single, double, and triple peak-pairs, respectively. Each peak-pair has unique 93 bp-long (±2 bp) ArgR-binding sequence containing two ARG boxes (39 bp) and residual sequence. Moreover, the peak-pairs provided the three ArgR-binding modes defined by the position of the two ARG boxes, indicating that the formation of DNA bending apparently centered between the pair of ARG boxes facilitates the non-specific contacts between ArgR subunits and the residual sequences. Thus, our data postulate the in vivo architecture of ArgR-DNA complexes to understand its transcription regulatory mechanism.
!Series_overall_design = ChIP-exo profiles of ArgR (+Arginine) and ArgR (-Arginine) were generated by deep sequencing in duplicates using Illumina MiSeq.
!Series_type = Genome binding/occupancy profiling by high throughput sequencing
!Series_contributor = Suhyung,,Cho
!Series_contributor = Yoo-Bok,,Cho
!Series_contributor = Haeji,,Yum
!Series_contributor = Taek-Jin,,Kang
!Series_contributor = Sun,C,Kim
!Series_contributor = Byung-Kwan,,Cho
!Series_sample_id = GSM1482120
!Series_sample_id = GSM1482121
!Series_contact_name = Byung-Kwan,,Cho
!Series_contact_laboratory = Systems and Synthetic Biology Lab
!Series_contact_institute = KAIST
!Series_contact_address = 291 Daehak-ro
!Series_contact_city = Daejeon
!Series_contact_zip/postal_code = 305-701
!Series_contact_country = South Korea
!Series_supplementary_file = ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE60nnn/GSE60546/suppl/GSE60546_RAW.tar
!Series_platform_id = GPL17439
!Series_platform_taxid = 511145
!Series_sample_taxid = 511145
!Series_relation = BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA258521
!Series_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP045631
^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 = GSM1482120
!Sample_title = ArgR (+arg) rep1 and rep2
!Sample_geo_accession = GSM1482120
!Sample_status = Public on Apr 01 2015
!Sample_submission_date = Aug 20 2014
!Sample_last_update_date = Apr 01 2015
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = ArgR (+arg) rep1 and rep2
!Sample_organism_ch1 = <Orgn>Escherichia coli</Orgn> str. <Strain>K-12</Strain> substr. <Substrain>MG1655</Substrain>
!Sample_taxid_ch1 = 511145
!Sample_characteristics_ch1 = chip antibody: Anti-myc (9E10) (Santa Cruz, Dallas, TX)
!Sample_characteristics_ch1 = substrain: MG1655
!Sample_treatment_protocol_ch1 = The cultured cells were inoculated with 1:100 dilution into 50 mL of the fresh <Med>M9 medium</Med> containing <Supp>2 g/L glucose</Supp> in either the <Supp>presence or absence of 1 g/L arginine</Supp> and continued to culture at <Temp>37°C</Temp> until reaching an appropriate cell density (<OD>OD600 ≈ 0.5</OD>).
!Sample_growth_protocol_ch1 = All strains used are E. coli K-12 MG1655 and its derivatives. Glycerol stock of the E. coli strain was inoculated into 3 mL Luria broth supplemented with 150 μg kanamycin and cultured overnight at 37°C with constant agitation.
!Sample_molecule_ch1 = genomic DNA
!Sample_extract_protocol_ch1 = Cultured cells (50 mL) were cross-linked with 1% formaldehyde at room temperature for 30 min and added 2 mL of 2.5M glycine to quench the unused formaldehyde. After washing  three times with 50 mL of ice-cold Tris-buffered saline (TBS), the washed cells were resuspended in 0.5 mL of lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 μg/mL RNaseA, protease inhibitor cocktail and 1 kU Ready-Lyse lysozyme (Epicentre, Madison, WI) and incubated at 37oC for 30 min. The cells were then treated with 0.5 mL of 2×IP buffer (100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 2%(v/v) Triton X-100, and protease inhibitor cocktail), followed by incubation on ice for 30 min. The lysate was then sonicated in an ice bath using Sonic Dismembrator Model 500 (four times for 20 s each, output level, 2.5). Size distribution of the fragmented DNAs was confirmed using agarose gel electrophoresis (200-400 bp) after removing cell debris by centrifugation.
!Sample_extract_protocol_ch1 = The cross-linked DNA-ArgR complexes in the supernatant were then immunoprecipitated by adding 10 µL of Anti-myc (9E10) (Santa Cruz, Dallas, TX). For mock-IP control, 2 µg of normal mouse IgG (Santa Cruz) was added into the supernatant in parallel. They were then incubated overnight at 4oC with constant rotation. The cross-linked DNA-protein and antibody complexes were selectively captured by adding 50 µL of Dynabeads Pan Mouse IgG magnetic beads (Invitrogen, Grand Island, NY). Then, DNAs were end-polished using T4 DNA polymerase (NEB, Ipswich, MA), ligated with the annealed adaptor 1 (5’- Phospho-AACTGCCCCGGGTTGCTCTTCCGATCT and 5’- OH-AGATCGGAAGAGC-OH), nick-repaired using phi29 polymerase (NEB), and digested with λ exonuclease (NEB) as reported previously. Then, protein-DNA complexes were reverse-cross-linked by heating at 65°C overnight and proteins were degraded by 8 µg of protease K (Invitrogen). The purified DNAs were denatured at 95°C and extended by P1 primer (5’-OH-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT), further ligated with the annealed adaptor 2 (5’-OH-ACACTCTTTCCCTACACGACGCTCTTCCGATCT and 5’-OH-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAG). The ligated DNA products were purified using Qiagen PCR purification kit and were PCR-amplified by P2 primer (5’-OH-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and P3 primer (5’-OH-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT). The degenerative sequence (the underlined 6Ns) in the P3 primer indicates the index sequence for the Illumina next-generation sequencing (Illumina, San Diego, CA). The PCR-amplified DNA products were then loaded onto 2% agarose gel and extracted using QIAquick gel purification columns.
!Sample_description = ChIP-exo
!Sample_data_processing = Basecalls performed using CASAVA version 1.4
!Sample_data_processing = All sequencing reads were mapped to E. coli MG1655 reference genome (<Gversion>NC_000913</Gversion>) using CLC Genomics Workbench5 with the length fraction of 0.9 and the similarity of 0.99.
!Sample_data_processing = To capture target protein binding sites corresponding genomic position of mapped reads start position (MRSP) was counted and stored for visual inspection using in-house scripts.
!Sample_data_processing = Supplementary_files_format_and_content: gff file is generated by in-house script
!Sample_platform_id = GPL17439
!Sample_contact_name = Byung-Kwan,,Cho
!Sample_contact_laboratory = Systems and Synthetic Biology Lab
!Sample_contact_institute = KAIST
!Sample_contact_address = 291 Daehak-ro
!Sample_contact_city = Daejeon
!Sample_contact_zip/postal_code = 305-701
!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/SAMN02999460
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX683718
!Sample_supplementary_file_1 = ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1482nnn/GSM1482120/suppl/GSM1482120_argR_arg.gff.gz
!Sample_series_id = GSE60546
!Sample_data_row_count = 0
^SAMPLE = GSM1482121
!Sample_title = ArgR (-arg) rep1 and rep2
!Sample_geo_accession = GSM1482121
!Sample_status = Public on Apr 01 2015
!Sample_submission_date = Aug 20 2014
!Sample_last_update_date = Apr 01 2015
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = ArgR (-arg) rep1 and rep2
!Sample_organism_ch1 = <Orgn>Escherichia coli</Orgn> str. <Strain>K-12</Strain> substr. <Substrain>MG1655</Substrain>
!Sample_taxid_ch1 = 511145
!Sample_characteristics_ch1 = chip antibody: Anti-myc (9E10) (Santa Cruz, Dallas, TX)
!Sample_characteristics_ch1 = substrain: MG1655
!Sample_treatment_protocol_ch1 = The cultured cells were inoculated with 1:100 dilution into 50 mL of the fresh M9 medium containing 2 g/L glucose in either the presence or <Supp>absence of 1 g/L arginine</Supp> and continued to culture at 37°C until reaching an appropriate cell density (OD600 ≈ 0.5).
!Sample_growth_protocol_ch1 = All strains used are E. coli K-12 MG1655 and its derivatives. Glycerol stock of the E. coli strain was inoculated into 3 mL Luria broth supplemented with 150 μg kanamycin and cultured overnight at 37°C with constant agitation.
!Sample_molecule_ch1 = genomic DNA
!Sample_extract_protocol_ch1 = Cultured cells (50 mL) were cross-linked with 1% formaldehyde at room temperature for 30 min and added 2 mL of 2.5M glycine to quench the unused formaldehyde. After washing  three times with 50 mL of ice-cold Tris-buffered saline (TBS), the washed cells were resuspended in 0.5 mL of lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 μg/mL RNaseA, protease inhibitor cocktail and 1 kU Ready-Lyse lysozyme (Epicentre, Madison, WI) and incubated at 37oC for 30 min. The cells were then treated with 0.5 mL of 2×IP buffer (100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 2%(v/v) Triton X-100, and protease inhibitor cocktail), followed by incubation on ice for 30 min. The lysate was then sonicated in an ice bath using Sonic Dismembrator Model 500 (four times for 20 s each, output level, 2.5). Size distribution of the fragmented DNAs was confirmed using agarose gel electrophoresis (200-400 bp) after removing cell debris by centrifugation.
!Sample_extract_protocol_ch1 = The cross-linked DNA-ArgR complexes in the supernatant were then immunoprecipitated by adding 10 µL of Anti-myc (9E10) (Santa Cruz, Dallas, TX). For mock-IP control, 2 µg of normal mouse IgG (Santa Cruz) was added into the supernatant in parallel. They were then incubated overnight at 4oC with constant rotation. The cross-linked DNA-protein and antibody complexes were selectively captured by adding 50 µL of Dynabeads Pan Mouse IgG magnetic beads (Invitrogen, Grand Island, NY). Then, DNAs were end-polished using T4 DNA polymerase (NEB, Ipswich, MA), ligated with the annealed adaptor 1 (5’- Phospho-AACTGCCCCGGGTTGCTCTTCCGATCT and 5’- OH-AGATCGGAAGAGC-OH), nick-repaired using phi29 polymerase (NEB), and digested with λ exonuclease (NEB) as reported previously. Then, protein-DNA complexes were reverse-cross-linked by heating at 65°C overnight and proteins were degraded by 8 µg of protease K (Invitrogen). The purified DNAs were denatured at 95°C and extended by P1 primer (5’-OH-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT), further ligated with the annealed adaptor 2 (5’-OH-ACACTCTTTCCCTACACGACGCTCTTCCGATCT and 5’-OH-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAG). The ligated DNA products were purified using Qiagen PCR purification kit and were PCR-amplified by P2 primer (5’-OH-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and P3 primer (5’-OH-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT). The degenerative sequence (the underlined 6Ns) in the P3 primer indicates the index sequence for the Illumina next-generation sequencing (Illumina, San Diego, CA). The PCR-amplified DNA products were then loaded onto 2% agarose gel and extracted using QIAquick gel purification columns.
!Sample_description = ChIP-exo
!Sample_data_processing = Basecalls performed using CASAVA version 1.4
!Sample_data_processing = All sequencing reads were mapped to E. coli MG1655 reference genome (<Gversion>NC_000913</Gversion>) using CLC Genomics Workbench5 with the length fraction of 0.9 and the similarity of 0.99.
!Sample_data_processing = To capture target protein binding sites corresponding genomic position of mapped reads start position (MRSP) was counted and stored for visual inspection using in-house scripts.
!Sample_data_processing = Supplementary_files_format_and_content: gff file is generated by in-house script
!Sample_platform_id = GPL17439
!Sample_contact_name = Byung-Kwan,,Cho
!Sample_contact_laboratory = Systems and Synthetic Biology Lab
!Sample_contact_institute = KAIST
!Sample_contact_address = 291 Daehak-ro
!Sample_contact_city = Daejeon
!Sample_contact_zip/postal_code = 305-701
!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/SAMN02999461
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX683719
!Sample_supplementary_file_1 = ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1482nnn/GSM1482121/suppl/GSM1482121_argR_noarg.gff.gz
!Sample_series_id = GSE60546
!Sample_data_row_count = 0

</gse>