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| Status |
Public on May 26, 2024 |
| Title |
K562 cells DUBR TSS mutant 2, BBX TSS viewpoint, biol_rep 2 |
| Sample type |
SRA |
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| Source name |
Bone marrow
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone marrow
cell line: K562
cell type: Chronic Myelogenous Leukemia
genotype: DUBR TSS mutant
treatment: None
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million cells were crosslinked with 2% formaldehyde for 10 minutes, followed by 5-minute quenching with glycine. Cells were washed once with PBS, and then cells were incubated for 20 min in lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal) with 1x protease inhibitors. For the first digestion, three rounds of digestion were made with DpnII (NEB R0543S) at 37 C in a thermomixer at 500 RPM (100U for 4 hours, 100U overnight, and 100U for another 4 hours); the restriction enzyme was inactivated by heating to 62 C for 20 minutes while shaking at 500 rpm. Then the first ligation was made with 2000U of T4 DNA ligase (NEB M0202L) at 16 C in 7mL of Milli-Q water overnight. Samples were then phenol-chloroform extracted and ethanol precipitated, and the second digestion was performed overnight with 50U of NlaIII (NEB R0125S) or HindIII (NEB R0104S). The second ligation was performed in 5mL total with 3000 units of T4 DNA ligase (NEB M0202L).
Libraries were constructed starting from the second ligation material which was purified with SPRI beads, and it was quantified with a Qubit dsDNA Assay Kit. A first round of PCR amplification with viewpoint-specific primers was performed with 4 x 50 µL PCR reactions with 200ng of 4C template using 16 PCR cycles and the Phusion polymerase (NEB M0530L). A second round of PCR with universal primers containing Illumina adapters was performed, and the material was purified with SPRI beads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
*library strategy: 4C-seq
Pipe4C was utilized for preprocessing and analysis of all samples
hg19 reference genome was used for all aligments
deeptools multiBamSummary was used to estimate scaling factors by viewpoint to create bigwig files using bamCoverage
Assembly: hg19
Supplementary files format and content: bigwig
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| Submission date |
Apr 23, 2024 |
| Last update date |
May 27, 2024 |
| Contact name |
Carlos Alberto Peralta-Alvarez |
| E-mail(s) |
cperalta@ifc.unam.mx
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| Organization name |
Universidad Nacional Autónoma de México
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| Department |
Genética Molecular
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| Lab |
122N
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| Street address |
Circuito Exterior s/n Ciudad Universitaria, Coyoacán
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| City |
Mexico City |
| State/province |
Ciudad de México |
| ZIP/Postal code |
03800 |
| Country |
Mexico |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE264663 |
A Bidirectional Non-Coding RNA Promoter Mediates Long-Range Gene Expression Regulation [4C-seq] |
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| Relations |
| BioSample |
SAMN41059682 |
| SRA |
SRX24345285 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8224967_bbx_m4_rep2_scaled.bigwig |
4.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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