Comment[ArrayExpressAccession]	E-GEOD-45242
MAGE-TAB Version	1.1														
Public Release Date	2013-10-23
Investigation Title	HoxD locus														
Comment[Submitted Name]	HoxD locus
Experiment Description	This SuperSeries is composed of the SubSeries listed below. Refer to individual Series														
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	LELEU														
Person First Name	Marion														
Person Email	geo@ncbi.nlm.nih.gov														
Person Affiliation	EPFL														
Person Address	School of Life Sciences, EPFL, EPFL-SV-ISREC-UPDUB- Station 19, Lausanne, Switzerland														
Person Roles	submitter														
Protocol Name	P-GSE45242-3	P-GSE45242-4	P-GSE45242-6	P-GSE45242-11	P-GSE45242-13	P-GSE45242-1	P-GSE45242-9	P-GSE45242-8	P-GSE45242-2	P-GSE45242-15	P-GSE45242-12	P-GSE45242-14	P-GSE45242-10	P-GSE45242-7	P-GSE45242-5
Protocol Description	standard Affymetrix protocol	standard Affymetrix protocol	Chromosome conformation capture on chip (4C) technique was performed as described (Hagège et al., 2007; Simonis et al., 2006). Caeca and brain were dissected from E13.5 embryos, dissociated with collagenase (Sigma Aldrich/Fluka) and filtered through a 35 micron mesh to isolate single cells. Cells were fixed in formaldehyde 2% for 10 min at room temperature and reaction was quenched on ice with glycine. Cells were further lysated with 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, PIC 1x to isolate nuclei. Nuclei from pools of one hundred caecum or two brains were digested with NlaIII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Sequences ligated to fragments of interest were digested again with DpnII (New England Biolabs), ligated with T4 DNA ligase HC (Promega) in diluted conditions and amplified using AmpliTaq DNA polymerase (Applied Biosystems) and inversed PCR primers flanked with adaptors allowing multiplexing. Hoxd13, Hoxd11, Hoxd9 and Hoxd4 PCR primers were described before (Noordermeer et al., 2011).	Polyadenylated RNA was prepared using TruSeq RNA Sample Preparation (Illumina).	Total RNA was depleted of rRNA using Ribo-Zero rRNA Removal Kit (Epicentre).	Chromosome conformation capture on chip (4C) technique was performed as described (Hagège et al., 2007; Simonis et al., 2006). Caeca were dissected from E13.5 embryos, dissociated with collagenase (Sigma Aldrich/Fluka) and filtered through a 35 micron mesh to isolate single cells. Cells were fixed in formaldehyde 2% for 10 min at room temperature and reaction was quenched on ice with glycine. Cells were further lysated with 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, PIC 1x to isolate nuclei. Nuclei from pools of fifty caecum or two brains were digested with DpnII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Sequences ligated to fragments of interest were amplified using previously described inversed PCR primers (Montavon et al., 2011) and AmpliTaq DNA polymerase (Applied Biosystems). 4C PCR was fragmented with apurinic / apyrimidinic endonucleases and uracil DNA glycosylases and labeled with terminal deoxynucleotidyl transferase using GeneChip WT Double- Stranded DNA Terminal Labeling Kit (Affymetrix). The profile of all different steps was analysed with a Bioanalyser. DNAs were hybridized to custom-made tiling arrays (Soshnikova and Duboule, 2009). Tiling arrays were processed according to manufacturer instructions.	RNA was depleted of rRNA 18S and 28S using RiboMinus Human/Mouse Transcriptome Isolation kit (Affymetrix). Using T7-(N)6 primers, RiboMinus-enriched RNA was stepwise reverse transcribed to cRNA (SuperScript II) and to double-stranded (ds) cDNA (DNA Polymerase I). cDNA was further amplifed by in vitro transcription to cRNA and reverse transcribed again to single-stranded (ss) cDNA (Superscript II) with incorporation of deoxyuridine using the GeneChip Whole Transcript Amplified Double-Stranded Target Assay kit (Affymetrix). For ss cDNA tiling array, cDNA was treated with NaOH 1M and blocked with HCl 1M. For ds cDNA tiling array, cDNA was reverse transcribed to ds cDNA (DNA Polymerase I) with incorporation of deoxyuridine. Both ss and ds cDNAs were fragmented with apurinic / apyrimidinic endonucleases and uracil DNA glycosylases and labeled with terminal deoxynucleotidyl transferase using GeneChip WT Double- Stranded DNA Terminal Labeling Kit (Affymetrix). The profile of all different steps was analysed with a Bioanalyser. cDNAs were hybridized to custom-made tiling arrays (Soshnikova and Duboule, 2009). Tiling arrays were processed according to manufacturer instructions. Transcript profiling experiment was repeated once for each sample, except for del(1-4i) and del(9-11) caeca.	ChIP was performed according to (Lee et al., 2006). Caeca were dissected from E13.5 embryos, fixed in formaldehyde 1% for 15 min at RT and washed three times with cold phosphate buffer solution (PBS). Pools of fifty caecum were sonicated with Bioruptor Next Gen (Diagenode), precleared with either EZview Red protein G/A Affinity Gel (Sigma) or Dynabeads M280 Sheep anti-Mouse IgG (Invitrogen) to avoid unspecific binding and immunoprecipitated with 2-6 ug of RNAPII (8WG16, Covance), H3K4me1 (ab8895, Abcam), anti-H3K4me3 (07-473, Millipore) or H3K27me3 (17-622, Millipore) antibodies and EZview Red protein G/A Affinity Gel (Sigma) / Dynabeads M280 Sheep anti-Mouse IgG (Invitrogen). After several washing steps, chromatin was eluted, decrosslinked and digested with RNAseA and Proteinase K. Immunoprecipitated chromatin and whole cell extract DNA (input) was blunted with T4 DNA Polymerase, and linker adaptors of oligos 1; 5’-GCGGTGACCCGGGAGATCTGAATTC-3’ and 2; 5’-GAATTCAGATC-3’ were ligated to blunt ends. This allowed amplification using ligation-mediated PCR (Lee et al., 2006). PCR was fragmented with apurinic / apyrimidinic endonucleases and uracil DNA glycosylases and labeled with terminal deoxynucleotidyl transferase using GeneChip WT Double- Stranded DNA Terminal Labeling Kit (Affymetrix). The profile of all different steps was analysed with a Bioanalyser. DNAs were hybridized to custom-made tiling arrays (Soshnikova and Duboule, 2009). Tiling arrays were processed according to manufacturer instructions. For each sample and antibody, ChIP-chip was repeated once.	Trizol	For transcription profiling along the hedgehog gut (E23), in addition to the stomach, the full-length intestine was separated into four isometric portions considered as the presumptive duodenum, jejunum, ileum and colon. Tissues were stored in RNAlater reagent (QIAGEN) until further processing. After disruption of single samples with PT1200E Polytron (Kinematica), RNA was isolated using either the RNeasy micro- or minikit (QIAGEN). Libraries and clusters were prepared and sequenced with Illumina Genome Analyzer IIx and paired-end sequencing of 100 bp.	For transcriptome profiling along the mouse gut (E13.5), in addition to stomach, caecum and colon samples, the small intestine was separated into three isometric portions considered as the presumptive duodenum, jejunum and ileum. Tissues were stored in RNAlater reagent (QIAGEN) until further processing. After disruption of single samples with PT1200E Polytron (Kinematica), RNA was isolated using either the RNeasy micro- or minikit (QIAGEN). Libraries and clusters were prepared and sequenced with Illumina Genome Analyzer IIx and paired-end sequencing of 100 bp.	Caeca were dissected from E13.5 embryos and and stored in RNAlater reagent (QIAGEN) until further processing. After disruption with PT1200E Polytron (Kinematica), RNA was isolated using the RNeasy minikit (QIAGEN). Libraries and clusters were prepared using standard Illumina protocols and sequenced with Illumina Genome Analyzer IIx and paired-end sequencing of 80 bp.	Caeca were dissected from E13.5 embryos and and stored in RNAlater reagent (QIAGEN) until genotyped. After disruption with PT1200E Polytron (Kinematica), RNA was isolated using the RNeasy minikit (QIAGEN).	4C PCR was sequenced with Illumina Genome Analyzer IIx. Data was mapped to the mouse genome using HTSstation (http://htsstation.vital-it.ch/).	standard Affymetrix protocol
Protocol Type	labelling protocol	hybridization protocol	sample treatment protocol	sample treatment protocol	sample treatment protocol	sample treatment protocol	sample treatment protocol	sample treatment 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	nucleic acid library construction protocol	array scanning protocol
Experimental Factor Name	SAMPLE TYPE	STRAIN OR LINE	CHIP ANTIBODY CATALOG #	CHIP ANTIBODY	CHIP ANTIBODY MANUFACTURER	GENOTYPE	AGE	ORGANISM	ORGANISM PART						
Experimental Factor Type	sample type	strain or line	chip antibody catalog #	chip antibody	chip antibody manufacturer	genotype	age	organism	organism part						
Publication Title	Multiple Enhancers Regulate Hoxd Genes and the Hotdog LncRNA during Cecum Budding.														
Publication Author List	Delpretti S, Montavon T, Leleu M, Joye E, Tzika A, Milinkovitch M, Duboule D														
PubMed ID	24075990														
Publication DOI	10.1016/j.celrep.2013.09.002														
Comment[SecondaryAccession]	GSE45242														
Comment[GEOReleaseDate]	2013-10-23														
Comment[ArrayExpressSubmissionDate]	2013-03-18														
Comment[GEOLastUpdateDate]	2013-10-23														
Comment[AEExperimentType]	other														
Comment[AEExperimentType]	ChIP-chip by tiling array														
Comment[AEExperimentType]	transcription profiling by tiling array														
Comment[AEExperimentType]	RNA-seq of coding RNA														
SDRF File	E-GEOD-45242.hyb.sdrf.txt	E-GEOD-45242.seq.sdrf.txt