|
| Status |
Public on May 08, 2024 |
| Title |
4C DKO rep1 |
| Sample type |
SRA |
| |
|
| Source name |
K562 cells with silencer1 and silencer2 DKO
|
| Organism |
Homo sapiens |
| Characteristics |
4c restriction enzyme: HindIII, DpnII
treatment: knockout silencer1 and 2 regions
cell line: K562
|
| Treatment protocol |
EV, S1KO and DKO cells undergoes the CRISPR excision.
|
| Growth protocol |
All the cells were cultured in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. All cultures were maintained at 37 °C, 5% CO2 in a humidified incubator.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
40 million cells were cross-linked with 1% formaldehyde. The nuclei pellets were isolated by cold lysis buffer (10mM Tris-HCl, 10mM NaCl, 5mM EDTA, 0.5% NP 40) supplemented with protease inhibitors (Roche). The first digestion was performed overnight at 37°C with HindIII enzyme (NEB). Digestion efficiency was measured by gel electrophoresis. After confirmation of good digestion efficiency, DNA was ligated overnight at 16°C by T4 DNA ligase (Thermo Scientific) and de-crosslinked by proteinase k. After that, DNA was extracted by phenol-chloroform and this DNA was referred as “3C library”. The “3C library” was then processed for second digestion with DpnII enzyme (NEB) overnight at 37°C. After ligation, we obtained “4C template DNA. The concentration of the 4C DNA was determined by Qubit assays (Thermo Scientific).
The 4C template DNA was then amplified using specific primers with Illumina Nextera adapters and sent for sequencing on the MiSeq platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Library strategy: 4C-seq
For 4C reads, we trimmed off the HINDIII digestion site using tagdust(2.33). The remaining read 1s were mapped by BOWTIE2(v2.2.5) with option '--end-to-end' in single-end mode. r3Cseq(1.24.0) was used to call interactions against the HINDIII digested genome background in hg19.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include interactions information from 4C-seq by r3cSeq
|
| |
|
| Submission date |
Jan 11, 2022 |
| Last update date |
May 08, 2024 |
| Contact name |
KAIJING CHEN |
| E-mail(s) |
KAIJING001@e.ntu.edu.sg
|
| Phone |
98667053
|
| Organization name |
Nanyang Technological University
|
| Department |
school of biological science
|
| Lab |
Melissa Fullwood
|
| Street address |
60 Nanyang Drive
|
| City |
Singapore |
| ZIP/Postal code |
637551 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE193483 |
Super-silencer perturbation by EZH2 and REST inhibition leads to large loss of chromatin interactions and reduction in cancer growth [4C-seq] |
| GSE193489 |
Super-silencer perturbation by EZH2 and REST inhibition leads to large loss of chromatin interactions and reduction in cancer growth |
|
| Relations |
| BioSample |
SAMN24848767 |
| SRA |
SRX13731839 |
