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Sample GSM4226445 Query DataSets for GSM4226445
Status Public on Sep 06, 2020
Title ChIPseq Hoxd9 in EB-Day3[D0+RA+SAG][D2+24hDox(iFlag.Hoxd9)] replicate 1
Sample type SRA
Source name ChIPseq Hoxd9 in EB-Day3[D0+RA+SAG][D2+24hDox(iFlag.Hoxd9)]
Organism Mus musculus
Characteristics strain genotype/variation: Ainv15(iFlag.Hoxd9)
treatment: EB + 3 Days RA + SAG, D2+24hours Dox
cell type: Embryoid Bodies
chip target: Hoxd9
chip antibody: Sigma, F1804
Treatment protocol Embryoid bodies (EBs) were obtained by plating trypsinized (Gibco) mESCs in AK medium (Advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), 7% KnockOut SR (vol/vol) (Gibco), 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol and penicillin–streptomycin (Gibco),) at 37 °C, 5% CO2 (day -2). On day 0, EBs were split 1:2 and AK medium was replenished and supplemented with 1 μM all-trans retinoic acid and 0.5 μM smoothened agonist (SAG) (Millipore, 566660). TF induction was performed by adding 3 μg/mL of Doxycycline (Sigma, D9891) on day 2. For ChIP-seq experiments, 3-3.5x10^6 mESCs were plated in 245mm x 245mm square dishes (Corning).
Growth protocol mESC lines were cultured in 2-inhibitors based medium (Advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), supplemented with 2.5% ESC-grade fetal bovine serum (vol/vol, Corning), N2 (Gibco), B27 (Gibco), 2mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1000 U/mL leukemia inhibitory factor (Millipore), 3μM CHIR (BioVision) and 1 μM PD0325901 (Sigma)) on 0.1% gelatin (Millipore) coated plates at 37 °C, 8% CO2.
Extracted molecule genomic DNA
Extraction protocol Cells were collected 24h and 48h after Doxycycline (Dox) treatment. Crosslinking was performed at room temperature in 1mM DSG (ProteoChem) for 15min, followed by the addition of 1% FA (vol/vol) for an additional 15min. After quenching with Glycine, cells were divided into ~25-30x106 aliquots, pelleted by centrifugation and frozen at -80°C. After thawing cells on ice, lysis was performed in 5mL of 50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol) with 1× protease inhibitors (Roche, 11697498001) for 10 min at 4 °C. Cells were centrifuged for 5min, resuspended in 5mL of 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 with 1× protease inhibitors, and incubated for 10 min at 4 °C on a rotating platform. Cells were centrifuged for 5min and resuspended in 2mL of Sonication Buffer (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100 (vol/vol), 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (vol/vol) with 1× protease inhibitors). Sonication was performed by splitting each sample to 2 Bioruptor tubes with sonication beads and using the Bioruptor Pico (Diagenode) for 18 cycles of 30 sec on and 30 sec off into an average size of approximately 200 bp. Immunoprecipitation was performed for 16h at 4 °C on a rotating platform by incubating with Dynabeads protein-G (Thermo Fisher Scientific) conjugated with 5 µg of one of the following antibodies: mouse monoclonal antibody to Flag (Sigma, F1804) or rabbit polyclonal antibody to HA (Abcam, ab9110).
lllumina DNA sequencing libraries were prepared with one third of the ChIP sample or a 1:100 dilution of the input sample in water. Library preparation was performed by end repair, A-tailing and ligating Illumina-compatible Bioo Scientific multiplexed adapters. Unligated adapters were removed using Agencourt AmpureXP beads (Beckman Coulter). Amplification was performed by PCR with Phusion polymerase (New England Biolabs) and TruSeq primers (Sigma). Libraries were gel purified (Qiagen) between 250 and 550bp in size. Final quantification of the library was performed using the KAPA library amplification kit on the Roche Lightcycler 480 before pooling.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Description HFJFLBGXC_n01_mb260
ES-derived embryoid bodies+3D RA+SAG with induced transcription factors
Data processing Basecall conversion to fastq files was performed using Picard (v 2.8.2)
Alignment: ChIP-seq reads were aligned to the mm10 genome using Bowtie (v1.0.1) (Langmead, Trapnell, Pop, & Salzberg, 2009), using options “-q --best --strata -m 1 --chunkmbs 1024”.
Binding Event Detection: Genome-wide TF binding events were called in each condition using MultiGPS (v0.74) (Mahony et al., 2014). EdgeR (v3.22.5) was used within the MultiGPS framework to test whether genomic sites were differentially bound by TFs (Robinson, McCarthy, & Smyth, 2010). A genomic site was defined as shared by TFs if significant binding events were called in both TF ChIP-seq experiments (q-value < 0.001), and the TFs did not display differential read enrichment at that site as estimated by EdgeR (q-value > 0.01). A binding event was defined as “TF1>TF2” if a MultiGPS peak was called in TF1 ChIP-seq (q-value < 0.001), TF1 exhibited a greater log fold-change with respect to the input ChIP-seq than TF2, and TF1 and TF2 were significantly differentially bound as defined by EdgeR (q-value < 0.01). A similar strategy was applied to perform multi-way ChIP-seq comparisons.
Genome_build: mm10
Supplementary_files_format_and_content: MultiGPS: MultiGPS output files
Supplementary_files_format_and_content: BED: Conversion of MultiGPS output files to BED format, centered on the MultiGPS binding event location
Submission date Dec 19, 2019
Last update date Sep 07, 2020
Contact name Shaun Mahony
Phone 814-865-3008
Organization name Penn State University
Department Biochemistry & Molecular Biology
Lab Shaun Mahony
Street address 404 South Frear Bldg
City University Park
State/province PA
ZIP/Postal code 16802
Country USA
Platform ID GPL19057
Series (2)
GSE142377 Diversification of posterior Hox patterning by graded ability to engage inaccessible chromatin [ChIP-seq]
GSE142379 Diversification of posterior Hox patterning by graded ability to engage inaccessible chromatin
BioSample SAMN13635395
SRA SRX7416187

Supplementary file Size Download File type/resource
GSM4226445_Day3_iHox_Hoxd9.bed.gz 509.7 Kb (ftp)(http) BED 1.3 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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