Comment[ArrayExpressAccession] E-GEOD-44756 MAGE-TAB Version 1.1 Public Release Date 2013-03-01 Investigation Title Hedgehog Molecular Networks in the Second Heart Field Comment[Submitted Name] Hedgehog Molecular Networks in the Second Heart Field 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 Yang Yang Hoffmann Moskowitz Person First Name Xinan Xinan Andrew Ivan Person Mid Initials "Holly" H D P Person Email xyang2@uchicago.edu Person Affiliation universty of Chicago Person Phone 773-702-5960 Person Address universty of Chicago, KCBD5121, 900 E. 57th Str., Chicago, IL, USA Person Roles submitter Protocol Name P-GSE44756-2 P-GSE44756-1 P-GSE44756-6 P-GSE44756-14 P-GSE44756-3 P-GSE44756-10 P-GSE44756-16 P-GSE44756-8 P-GSE44756-7 P-GSE44756-13 P-GSE44756-9 P-GSE44756-15 P-GSE44756-11 P-GSE44756-4 P-GSE44756-12 P-GSE44756-5 Protocol Description ID_REF = VALUE = Variance Stabilizing Normalization (VSN) normalized signal intensity in log2 scale ID_REF = VALUE = Variance Stabilizing Normalization (VSN ) normalized signal intensity on log2 scale 1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60M-0C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4112F) for 17 hours at 65M-CM-^BM-0C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37M-CM-^BM-0C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation. Mef2c-AHF-Cre transgenic mice and homozygous Rosa26Gli3TFlag mice (http://jaxmice.jax.org/strain/013124.html) were crossed and dissected at E10.5. SHF tissue was isolated from these animals individually, and litters were pooled in groups of approximately 50 embryos. Mice were raised in the University of Chicago's Animal Resource Care facilities. Tbx5 -/+ mice were maintained on a mixed background. Female mice were administered a combination of pregnant mare serum gonadotropin and human chorionic gonadotropin prior to mating to induce large litters. Embryos were dissected from pregnant female mice at 9.5 days post fertilization in cold, RNase-free PBS. SHF tissues were immediately isolated. To get the Shh -/- mice, shh +/- female mice on mixed background were superovulated using 5 units each pregnant mare serum gonadotropin (PMS-G) and human chorionic gonadotropin (HCG) administered 48 hours apart. They were mated to Shh +/- male mice after HCG administration. Embryos were isolated at E9.5. The reads of Gli3 IP is 44bp while the reads of the Input is 36bp. We trimed the left 8bp of the Gli3 IP due to their unexpected low quality scores. ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie version 0.12.8 allowing 2 mismatches and assuming genome_size to be 2e9. Peaks were called using MACS version 2.0.7 with the following setting: ChIP threshold (p<1e-5), m Fold (2,100), band width (200). The peaks of GLI3 IP was compared with the peaks of Input. Genome_build: mm9 Supplementary_files_format_and_content: Gli3 binding sites were annotated to the mouse mm9 reference genome by HOMER software, by the nearst transcription start sites (TSSs). The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 014868_D_F_20090416) to obtain median background subtracted mean signal of the spot. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Slides were scanned immediately after washing on the Agilent Technologies Scanner (G2505C US80900103) using one color scan setting for 4x44k array slides (Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%). Slides were scanned immediately after washing on the Agilent Technologies Scanner (G2505C US80900103) using one color scan setting for 4x44k array slides (Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%). Posterior and anterior second heart field (SHF) tissues were isolated from each embryo and stored in RNAlater RNA stabilization reagent (QIAGEN) at 4 M-0C. Non-cardiac tissues were saved for genotyping. After genotyping, Shh -/- and Shh +/+ embryos were pooled, homogenized and RNA extracted. Chromatin was fixed in 1.8% formaldehyde solution for 10 minutes and sonicated to 300-500 bp fragments using a Misonex 4000 sonicator. Chromatin was immunoprecipitated overnight using a pre-cleared anti-FlagM2 anitbody (Sigma) and magnetic beads (invitrogen). Chromatin was eluted, de-crosslinked and column-purified using a PCR cleanup kit (Qiagen) before submission for sequencing. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3M-bM-^@M-^Y end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. RNA was extracted from pooled, homogenized SHF tissues using the QIAGEN RNeasy kit following the manufacturer's recommendations. The protocol includes an on-column DNase treatment. RNA was quantified using a Nanodrop ND-1000 spectrometer and quality was checked using an agilent bioanalyzer. RNA was prepared using the RNAeasy RNA Purification kit (Qiagen) following the manufacturer's recommendations (September 2010 version). The protocol includes homogenization of the tissue fragments and an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Cy3 labeled cRNA was created using the agilent quick-amp RNA labeling kit with RNA spike-in, followed by purification using a QIAGEN RNeasy kit. 0.5 ug was used for each sample, and the labeling was validated using a nanodrop spectrometer. Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. Protocol Type bioassay_data_transformation bioassay_data_transformation hybridization grow grow grow feature_extraction feature_extraction image_aquisition image_aquisition specified_biomaterial_action nucleic acid library construction protocol nucleic acid library construction protocol nucleic acid library construction protocol labeling labeling Experimental Factor Name GENOTYPE STRAIN BACKGROUND ORGANISM PART Experimental Factor Type genotype strain background organism part Comment[SecondaryAccession] GSE44756 Comment[GEOReleaseDate] 2013-03-01 Comment[ArrayExpressSubmissionDate] 2013-02-28 Comment[GEOLastUpdateDate] 2013-03-03 Comment[AEExperimentType] ChIP-seq Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-44756.hyb.sdrf.txt E-GEOD-44756.seq.sdrf.txt