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| Status |
Public on Aug 30, 2020 |
| Title |
ENHIC11; Hi-C_CTCF-AID_TetO-CTCF(Del_13-33)_auxin+dox2days_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
mouse ES cells, line EN281.1
|
| Organism |
Mus musculus |
| Characteristics |
genotype: CTCF-AID,Tir1_Rtta3G-TetO-CTCF(Del_13-33) clone 1
treatment: auxin+dox2days
|
| 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. Doxycycline was added at 1ug/ml final from a 1mg/ml stock.
|
| 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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq and Hi-C cells were trypsinized and fixed in suspension; For 5C cells were fixed in tissue culture plates and scrapped off
ChIP libraries were prepared using standard Illumina protocols;5C (in-situ ligation with HindIII) and Hi-C (dilution ligation, Arima genomics kit) were assembled based on Illumina-compatible protocols. Detailed experimental procedures can be found in the supplementary information of the linked manuscript
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| |
|
| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
ChIP-seq. 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. CTCF (FLAG) peaks were called using the geome wide Event finding and Motif Discovery (GEM) algorithm.
We processed each Hi-C dataset using distiller (https://github.com/mirnylab/distiller, doi:10.5281/zenodo.3350937), mapping reads to mm10 and saving processed data in the cooler format.
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 for ChIP-seq
Genome_build: custon 5C genome for 5C (provided as supplementary information) positions referring to mm9
Genome_build: mm10 for Hi-C
Supplementary_files_format_and_content: Hi-C .cool: valid Hi-C pairs mapped at 10kb resolution
Supplementary_files_format_and_content: ChIP-seq .bed: position of the CTCF (FLAG) peaks.
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 filtered symmetrical read-normalized balanced (Iterative Correction method - ICE) matrices.
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| |
|
| Submission date |
Aug 25, 2020 |
| Last update date |
Aug 30, 2020 |
| 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) |
| GSE156868 |
Molecular basis of CTCF binding polarity in genome folding |
|
| Relations |
| BioSample |
SAMN15939320 |
| SRA |
SRX9034667 |
