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| Status |
Public on Apr 29, 2021 |
| Title |
A673_siSA2-Dh8_72h_HiChIP_CTCF_Rep2_Ad D347C082 |
| Sample type |
SRA |
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| Source name |
Ewing sarcoma cell line (A673)
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| Organism |
Homo sapiens |
| Characteristics |
sirna: siSA2-Dh8
antibody: CTCF (provided in iDeal ChIP-seq kit Diagenode)
timepoint: 72h
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| Treatment protocol |
Cells were transfected for 72h with either siCT, siSA2#6 or siSA6#8 (Dharmacon). For each siRNA, CTCF HiChIP experiments were preformed. Several adaptations were made and are detailed below in an overview of the otherwise original protocol described by Mumbach and colleagues (PMID: 27643841). Cells were fixed at room temperature for 10 min under gentle shaking platform (50mvt/min). 2 mL of glycine solution (2M) were added and cells were incubated for 5 additional min at room temperature under gentle shaking platform. The supernatant was removed and cells were washed 3 times with PBS at room temperature. Cells were scraped vigorously with ice cold PBS supplemented with protease inhibitor cocktail tablets (11836145001, Roche), and flushed five time through a syringe with a 21 gauge needle (301155, BD Microlance). In situ contact libraries were performed starting from 15 million nuclei digested overnight at 37°C with Mbo1 (R0147M, New England Biolabs). After proximity ligation (4 h at room temperature), the nuclear pellet was sonicated and the chromatin immunoprecipitation step was performed using the iDeal ChIP-seq kit for transcription factors (Diagenode) according to the supplier's recommendation with some modification. Nuclei were resuspended in IL1b and IL2 buffer following Diagenode protocol and all centrifugations were performed at 4°C for 5 min at 1,950 RCF for these steps. The chromatin was sonicated (5 million nuclei per tube) with Bioruptor pico (Diagenode) for 10 cycles (30-sec on, 30-sec off). The tubes were pooled and the chromatin was clarified by centrifugation at 4°C for 10 min at 16,000 RCF. Sonicated chromatin from 11 million nuclei were used for CTCF immunoprecipitation step using the equivalent of 3 ChIP reactions pooled in one tube for each HiChIP reaction (final volume was 1050 µl/tube). Immunoprecipitation was then carried out following Diagenode kit instructions by multiplying all reagents by a factor 3 until end of elution step (50µl). Biotin capture was performed following Mumbach et al. protocol (PMID: 27643841).
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| Growth protocol |
A673 were cultured in DMEM supplemented with 10% fetal bovine serum. Four days before performing the ChIP experiments, cells were seeded in T150 flask at a concentration allowing to reach a cellular confluence of approximately 90% at the time of fixation of the cells with formaldehyde.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Library for Illumina sequencing were prepared using 10 ng of chromatin and 0.5 µL of TN5 (15028211, Illumina). A first PCR in a final volume of 50µl with 5 cycles was performed (72 °C for 5 min, 98 °C for 1 min, then 5 cycles at 98 °C for 15 s, 63 °C for 30 s, and 72 °C for 1 min). To determine how many additional PCR cycles were required for optimal library preparation, a QPCR with: 5µL of the first PCR product, 1 μL of Nextera Ad1_noMX and Nextera Ad2.X (each at 1.25 μM), 5 µl of phusion HF 2X (M0531S, New England Biolabs), 0,75 µL Evagreen® 20X (31000, Biotium) and 2,25 µL of water was runned (same program as first PCR but with 30 cycles). The optimal number of additional cycle was determined for each library by setting a threshold just before reaching the end of exponential amplification step. The 45µl left from the first PCR were further amplified using additional PCR cycles as determined above. Size selection was performed using Ampure XP beads (A63881, Beckman Coulter) to capture fragments greater than 300 bp. Libraries were quantified and analyzed using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and LabChIP (Perkin Elmer). A first validation of HiChIP experiments was performed using 150 bp paired-end sequencing on MiSeq-microV2-300-PE150 (Illumina). Deep 101 bp paired-end sequencing was then performed on NovaSeq 6000 systems (Illumina).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
A673_siSA2-Dh8_72h_HiChIP_CTCF_Rep2_Ad
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| Data processing |
All the processing steps were performed with HiC-Pro 2.10.1 pipeline.
Reads were mapped against human genome (GRCh37/hg19 version) with bowtie2.
Default HiC-Pro settings were used to remove duplicate reads, assign reads to MboI restriction fragments, filter for valid interactions and generate raw interaction matrices (5kb bins).
Genome_build: hg19
Supplementary_files_format_and_content: Raw interaction matrix (for each 5kb bin, count of valid pairs)
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| Submission date |
Aug 21, 2020 |
| Last update date |
Apr 29, 2021 |
| Contact name |
Olivier Delattre |
| Organization name |
Institut Curie
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| Department |
U830
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| Lab |
Olivier Delattre
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| Street address |
26 rue d'ulm
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| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE133228 |
STAG2 promotes CTCF-anchored loop extrusion and cis-promoter and -enhancer interactions |
| GSE156650 |
CTCF HiChIP data in A673 Ewing sarcoma cell lines (siCT & siSA2) |
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| Relations |
| BioSample |
SAMN15878132 |
| SRA |
SRX8984346 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4735782_D347C082_5000.matrix.gz |
20.0 Mb |
(ftp)(http) |
MATRIX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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