Comment[ArrayExpressAccession] E-GEOD-43504 MAGE-TAB Version 1.1 Public Release Date 2013-02-04 Investigation Title Genome-wide mapping of early replication fragile sites (ERFS) Comment[Submitted Name] Genome-wide mapping of early replication fragile sites (ERFS) Experiment Description DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins RPA, SMC5, gamma-H2AX, and BRCA1 in B cells subjected to replication stress. Protein-DNA association for four DNA damage response proteins (RPA, SMC5, g-H2AX, BRCA1), BrdU incorporation, and gene transcription in B lymphocytes with and without hydroxyurea treatment were examined. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Faryabi Faryabi Barlow Person First Name Robert Robert Jacqueline Person Mid Initials Babak B H Person Email faryabib@mail.nih.gov Person Affiliation National Institutes of Health Person Phone 301-435-6485 Person Address National Cancer Institutes, National Institutes of Health, 9000 Rockville Pike, Building 37, Bethesda, MD, USA Person Roles submitter Protocol Name P-GSE43504-10 P-GSE43504-3 P-GSE43504-11 P-GSE43504-9 P-GSE43504-7 P-GSE43504-2 P-GSE43504-8 P-GSE43504-4 P-GSE43504-6 P-GSE43504-1 P-GSE43504-5 Protocol Description Illumina Casava1.7.0 or 1.8.0 software used for basecalling. RNA-Seq sequenced reads were mapped to mm9 using Tophat v2.0.6. Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using exons of each transcript annotated by BioConductor's GenomicFeatures 1.10.1. Genome_build: mm9 Illumina Casava1.7.0 or 1.8.0 software used for basecalling. Sequenced reads were trimmed, and masked for low-quality sequence, then mapped to mm9 whole genome using bowtie v0.12.8 determined uniquely aligned reads with at most 2 mismatches. SICER with parameters: e-value, 100, gap size 600 (200) for RPA and γ−H2AX (BRCA1 and SMC5) is used to find enriched bins over Poisson distribution. Then the enrichment of bins passing the first criterion was examined relative to whole-cell extract control based on FDR of 1x10-5. Subsequently, the identified enriched windows that were less than 5 kbp apart were merged to delineate islands. Genome_build: mm9 Illumina Casava1.7.0 or 1.8.0 software used for basecalling. Genome_build: mm9 RNA-Seq sequenced reads were mapped to mm9 using Tophat v2.0.6. Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using exons of each transcript annotated by BioConductor's GenomicFeatures 1.10.1. Total RNA was isolated using TRIzol (Ambion) following manufacturer's protocol. RNA was washed, purified using RNeasy kit (Qiagen), and measured for quality. RNA was then prepared for sequencing using the TruSeq RNA sample prep kit (Illumina). Four libraries were sequenced on one lane of HiSeq 2000. Illumina sequencing libraries were constructed from the random-primed BrdU-DNA material according to standard procedures according to Hansen et al. PNAS, 107, 139-144, 2010. Each library was sequenced on a Genome Analyzer IIx. Cells were fixed with 1% paraformaldehyde followed by quenching with 0.125 glycine and sonication. Debris was removed by centrifugation and the supernatant diluted 10-fold in immunoprecipitation buffer, protease inhibitors (EDTA-free, Roche), and phosphatase inhibitors (Cocktail II, Sigma) and incubated with antibodies overnight. The next morning, chromatin fragments were then immunoprecipitated with the antibodies shown in the sample characteristics following protocols described in the manuscript. Immunoprecipitates were processed following Illumina’s protocol and sequenced on a Genome Analyzer IIx. DNAse treated DNA was extracted and amplified according to Sekimata et al., Immunity, 31, 551-564, 2009. Library was sequenced on a Genome Analyzer IIx. Cells were fixed with 1% paraformaldehyde followed by quenching with 0.125 glycine and sonication. Chromatin fragments were then immunoprecipitated with the antibodies shown in the sample characteristics following protocols described in the manuscript. Immunoprecipitates were processed following Illumina’s protocol and sequenced on a Genome Analyzer IIx. CD43- resting B cells were isolated from mouse spleens by negative selection with magnetic beads (Miltenyi Biotech). Following isolation, resting B cells were stimulated with LPS (25 mcg/ml, Sigma) and IL-4 (5 ng/ml, Sigma) in media containing 10 mcM BrdU for 28 hrs. CD43- resting B cells were isolated from mouse spleens by negative selection with magnetic beads (Miltenyi Biotech). Following isolation, resting B cells were stimulated with LPS (25 mcg/ml, Sigma) and IL-4 (5 ng/ml, Sigma) for 28 hrs. CD43- resting B cells were isolated from mouse spleens by negative selection with magnetic beads (Miltenyi Biotech). Following isolation, resting B cells were stimulated with LPS (25 mcg/ml, Sigma) and IL-4 (5 ng/ml, Sigma) for 72 hrs. Protocol Type normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol nucleic acid library construction protocol nucleic acid library construction protocol nucleic acid library construction protocol nucleic acid library construction protocol nucleic acid library construction protocol growth protocol growth protocol growth protocol Experimental Factor Name TREATMENT PROTOCOL CHIP ANTIBODY GROWTH DURATION GENOTYPE IMMUNOPRECIPITATE Experimental Factor Type treatment protocol chip antibody growth duration genotype immunoprecipitate Publication Title Identification of early replicating fragile sites that contribute to genome instability. Publication Author List Barlow JH, Faryabi RB, Call�n E, Wong N, Malhowski A, Chen HT, Gutierrez-Cruz G, Sun HW, McKinnon P, Wright G, Casellas R, Robbiani DF, Staudt L, Fernandez-Capetillo O, Nussenzweig A PubMed ID 23352430 Publication DOI 10.1016/j.cell.2013.01.006 Comment[SecondaryAccession] GSE43504 Comment[GEOReleaseDate] 2013-02-04 Comment[ArrayExpressSubmissionDate] 2013-01-15 Comment[GEOLastUpdateDate] 2013-07-23 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AEExperimentType] ChIP-seq Comment[AdditionalFile:Data1] GSE43504_HU_WT_Brdu.island.bed Comment[AdditionalFile:Data2] GSE43504_torpkm_RNASeq_byExon.txt Comment[SecondaryAccession] SRP017951 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR648771-SRR648794 SDRF File E-GEOD-43504.sdrf.txt