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Status |
Public on Jan 06, 2020 |
Title |
CTCF ChIP - CTCF Y226A, F228A |
Sample type |
SRA |
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Source name |
CTCF Y226A, F228A
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Organism |
Homo sapiens |
Characteristics |
cell line: HAP1 genotype: CTCF Y226A, F228A replicate: 1 chip antibody: CTCF (Cell Signaling 3418S)
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Treatment protocol |
gRNA targeting exon 1 of CTCF were designed and annealed into pX330 (primer: 5’- CGATTTTGAGGAAGAACAGC-3’). To modify the targeted locus we co-transfected a 120 basepair repair oligo containing the desired mutation and a silent mutation (repair oligo: 5’ – CCAAAAAGAGCAAACTGCGTTATACAGAGGAGGGCAAAGATGTAGATGTGTCTGTCGCCGATGCTGAAGAAGAACAGCAGGAGGGTCTGCTATCAGAGGTTAATGCAGAGAAAGTGGTTG-3’). pBabePuro was co-transfected in a 10:1 ratio to the pX330. Transfected clones were selected using 2 µg/µl puromycin for 2 days. gDNA of clones was isolated and send for Sanger sequencing to check for the desired mutation.
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Growth protocol |
HAP1 cells (Carette et al., 2011) were cultured in IMDM (Invitrogen) supplemented with 10% FCS (Clontech), 1% Penicillin-Streptomycin (Invitrogen) and 0.5% Ultraglutamin (Lonza).
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were fixed in 1% formaldehyde; 50mM Hepes-KOH; 100mM NaCl; 1 mM EDTA; 0,5 mM EGTA. Cell lysis was performed in LB1 buffer with final pH 7,5 (50 mM Hepes-KOH; 140mM NaCl; 1mM EDTA, 10% Glycerol, 0.5% NP-40; 0,25% Triton X-100; Proteinase inhibitor) for 20 minutes at 4°C. Subsequently, nuclei were lysed using LB2, pH 8,0 (10mM Tris-HCl; 200 mM NaCl; 1mM EDTA; 0,5mM EGTA; Proteinase inhibitor) for 10 minutes at 4°C. Pellets were resuspended in LB3 pH8,0 (10mM Tris-HCl; 100mM NaCl; 1 mM EDTA; 0,5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine; Proteinase inhibitor). Cross-linked chromatin was sheared (400-800 bp) using a Covaris S2 with Tube and Caps (Covaris, 520048), using the following settings: duty cycle: 10%, intensity: 4, cycles per burst: 200 time 40 seconds with 20 cycles. Chromatin precipitation was performed overnight using antibody-bound (CTCF 5 μl, SMC1 10 μl per IP) proteinA coupled DynaBeads (Invitrogen). Elution and decrosslinking was performed overnight at 65°C in EB pH 8,0 (50mM Tris-HCl; 10mM EDTA; 1% SDS). Samples were treated with Proteinase K and RNAseA for 2 hours. DNA was isolated using phenol extraction and ethanol precipitation. Library preparation was done using a KAPA Library preparation kit using the manufacturer’s protocol.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Reads were trimmed using TrimGalore (Martin, 2011). Reads were mapped to hg19 using Bowtie2.1.0 (Langmead and Salzberg, 2012) with default settings. Peak calling was performed using MACS2 (Feng et al., 2012) with default options. Genome_build: hg19 Supplementary_files_format_and_content: narrowPeak
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Submission date |
Aug 29, 2019 |
Last update date |
Sep 10, 2020 |
Contact name |
Angela Sedeno Cacciatore |
E-mail(s) |
a.sedeno.cacciatore@nki.nl, m.v.ruiten@nki.nl
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Organization name |
Netherlands Cancer Institute
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Department |
Gene regulation
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Lab |
Rowland lab
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Street address |
Plesmanlaan 121
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City |
Amsterdam |
State/province |
Noord-Holland |
ZIP/Postal code |
1066 CX |
Country |
Netherlands |
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Platform ID |
GPL16791 |
Series (2) |
GSE126634 |
The structural basis for cohesin/CTCF anchored loops [ChIP-seq] |
GSE126637 |
The structural basis for cohesin/CTCF anchored loops |
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Relations |
BioSample |
SAMN12657630 |
SRA |
SRX6777954 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4052953_CTCF_ChIPseq_CTCF.Y226A.F228A_peaks.narrowPeak.gz |
2.2 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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