 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 25, 2017 |
| Title |
CTCF_ChIP-seq_CTCF-AID_untreated_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
mouse ES cells E14Tg2a
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129/Ola, XY
genotype: CTCF-AID, Tir1
chip epitope: CTCF (Active Motif 61311) + Spike in antibody (Active Motif 61686)
|
| Treatment protocol |
For induction of the auxin-inducible degron indole-3-acetic acid (IAA, chemical analog of auxin) was added in the medium at 500µM from a 1000X stock diluted in sterile water and kept at 4°C up to 4 weeks or -20°C for long term storage.
|
| Growth protocol |
E14Tg2a (karyotype 19, XY; 129/Ola isogenic background) and subclones were cultured in DMEM+Glutamax (ThermoFisher cat 10566-016) supplemented with 15% Fetal Bovine Serum (ThermoFisher SH30071.03), 550µM b-mercaptoethanol (ThermoFisher 21985-023), 1mM Sodium Pyruvate (ThermoFisher 11360-070), 1X non-essential amino-acids (ThermoFisher 11140-50) and 104U of Leukemia inhibitory factor (Millipore ESG1107). Cells were maintained at a density of 0.2-1.5x105 cells / cm² by passaging using TrypLE (12563011) every 24-48h on 0.1% gelatin-coated dishes (Millipore cat ES-006-B) at 37°C and 7% CO2. Medium was changed daily when cells were not passaged. Cells were checked for mycoplasma infection every 3-4 months and tested negative. mouse ES cells were targeted to insert an Auxin-Inducible Degron (AID) tag together with an eGFP cassette, and the auxin receptor OsTir1 was expressed from a transgene. ChIP-seq, Hi-C, 5C and RNA-seq were performed at different timepoints after adding auxin to the culture medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq cells were trypsinized and fixed in suspension; For Chromosome Conformation Capture cells were fixed in tissue culture plates and scrapped off; For RNA-seq cells werre washed once with PBS and lysed in Trizol in the tissue culture plate.
ChIP libraries were prepared using standard Illumina protocols; RNA-seq libraries were assembled using the non-stranded kit E7530L from NEB; 5C (in-situ ligation) and Hi-C (dilution ligation) were assembled based on Illumina-compatible protocols. Detailed experimental procedures can be found in the supplementary information of Nora et al. 2017 Cell
ChIP-seq, ChIP-exo, RNA-seq, Hi-C, 5C
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIP-seq
|
| Data processing |
ChIP-seq and ChIP-exo. Fastq files were trimmed using the fastq-mcf program, aligned to the mm9 reference genome with bowtie2 (Langmead and Salzberg, 2012). Reads with a mapq score of 30 or greater were retained, using Samtools. PCR duplicates were filtered out. for CTCF peaks were called using the geome wide Event finding and Motif Discovery (GEM) algorithm. For H3K27me3 peaks that are broader we used a threshold-based method.
RNA-seq. Alignment was produced using STAR version 2.5.0a , aligned to mm9. Cufflinks version 2.2.1 with Bedtools version 2.17.0 were used as to produce input for differential expression analysis with Cuffdiff . Genes with an FPKM below 1.1 in all conditions were not considered in the differential expression analysis
Hi-C. We mapped the sequence of Hi-C molecules to reference mouse genome assembly mm9 using Bowtie 2.2.8 and the iterative mapping strategy, as described in (Imakaev et al., 2012; Lajoie et al., 2015). Upon filtering PCR duplicates and reads mapped to multiple or zero locations, we aggregated the reads pairs into 20kb and 100kb genomic bins to produce Hi-C contact matrices. To remove the short-range Hi-C artifacts - unligated and self-ligated Hi-C molecules - we ignored the contacts mapping to the same or adjacent genomic bins in all downstream analyses.
5C. Adapters were trimmed and aligment was performed using Bowtie2 against a pseudo-genome composed of all possible Forward-Reverse pairs of 5C oligonucleotides and matrices were analyzed using my5C (Lajoie et al. 2009 Nature Methods).
Genome_build: mm9
Supplementary_files_format_and_content: 5C - for all forward-reverse possible ligation pair we provide one file with raw 5C counts (not read normalized, including anchors ultimately removed from analysis), and one file with anchor filtered symmetrical read-normalized balanced (Iterative Correction method - ICE) matrices.
Supplementary_files_format_and_content: ChIP-seq and ChIP-exo. _Peaks.bed: position of the peaks. _Footprints.bed: position of ChIP-exo footprint. _tagDensity.bw: ChIP signal.
Supplementary_files_format_and_content: RNA-seq. .bw: tagDensity of signal.
|
| |
|
| Submission date |
May 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Elphege Nora |
| Organization name |
University of California San Francisco
|
| Department |
Cardiovascular Research Institute
|
| Lab |
Elphege Nora
|
| Street address |
555 Mission Bay Boulevard South
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE98671 |
Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization |
|
| Relations |
| BioSample |
SAMN06917463 |
| SRA |
SRX2790845 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2609185_CTCF_ChIP-seq_CTCF-AID_untreated_rep1_ENC124_Peaks.bed.gz |
432.8 Kb |
(ftp)(http) |
BED |
| GSM2609185_CTCF_ChIP-seq_CTCF-AID_untreated_rep1_ENC124_tagDensity.bw |
455.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
