 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 03, 2017 |
| Title |
DKO_3.3 SMC1 ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
Hap1 WaplKO_3.3/SCC4KO
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Hap1
genotype/variation: WaplKO_3.3/SCC4KO
antibody: SMC1
|
| Treatment protocol |
CRISPRs targeting WAPL (5’-CACCGCGTTCCATAGTATCCTGTA-3’) and SCC4 (5’-CACCGTACGGGCCTCGATGCGCTG-3’) were cloned into px330 (Addgene plasmid #42230). ∆WAPL and ∆SCC4 HAP1 clones were generated by insertion of a Blasticidine or Puromycin cassette respectively, as previously described (Blomen et al., 2015). ∆WAPL/∆SCC4 cells were generated by knocking out SCC4 in ∆WAPL cells.
|
| Growth protocol |
HAP1 cells (Carette et al., 2011) were cultured in IMDM (Invitrogen) supplemented with 10% FCS (Clontech), 1% Penicillin-Streptomycin (Invitrogen) and 1% Ultraglutamin (Lonza).
|
| 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, Igg 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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Reads were mapped to hg19 using Bowtie2.1.0 with default settings.
Peak calling was performed using MACS2 (v2.1.1) (Feng et al., 2012) using IgG as control.
Genome_build: hg19
Supplementary_files_format_and_content: narrowPeak
|
| |
|
| Submission date |
Feb 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Robin H. van der Weide |
| Organization name |
Hubrecht Institute
|
| Lab |
Kind Lab
|
| Street address |
Uppsalalaan 8
|
| City |
Utrecht |
| State/province |
Utrecht |
| ZIP/Postal code |
3584 CT |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE94992 |
The cohesin release factor WAPL restricts chromatin loop extension. [ChIP-Seq] |
| GSE95015 |
The cohesin release factor WAPL restricts chromatin loop extension. |
|
| Relations |
| BioSample |
SAMN06344483 |
| SRA |
SRX2570615 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2493885_3885_16_SMC1_DKO_CCGTCC_S19_L004_R1_001_peaks.narrowPeak.gz |
712.9 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
