Illumina HiSeq 2000 sequencing; A deconvolution protocol for chip-seq reveals analogous enhancer structures on the mouse and human ribosomal RNA genes
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Blood
Cell type
K-562
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Chronic Myelogenous
Attributes by original data submitter
Sample
ENA first public
2017-12-31
ENA last update
2017-08-31
ENA-CHECKLIST
ERC000011
External Id
SAMEA104233034
INSDC center alias
Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, and Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University
INSDC center name
Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, and Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University
INSDC first public
2017-12-31T17:01:44Z
INSDC last update
2017-08-31T10:25:37Z
INSDC status
public
Submitter Id
E-MTAB-6032:Sample 2
broker name
ArrayExpress
cell line
K562
cell type
lymphoblast
common name
human
disease
chronic myelogenous leukemia
organism part
bone marrow
sample name
E-MTAB-6032:Sample 2
Sequenced DNA Library
library_name
Sample 2_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed in 1% formaldehyde for 8 min at room temperature. Nuclei were isolated using Lysis Buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40), transferred to Sonication Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EGTA, 4 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% NP-40) and sonicated (Bioruptor, Diagenode) for 30 cycles of 30 sec on / 30 sec off at high intensity. Each immunoprecipitation (IP) was carried out on the equivalent of 50 x 106 cells in IP Buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100) overnight at 4°C. The antibody slurry was prepared with 50 µl A-, 50 µl G-Dynabeads and 60 µg.ml-1 antibody per IP. See; Herdman et al. (2017). PLoS Genetics 13, e1006899. Rabbit polyclonal antibodies against mouse UBF, RPI large subunit (RPA194/Polr1A)were generated in the laboratory. Anti-RPA194 (#5) and anti-UBF (#8) antisera were raised against mouse RPA194 (aa 231–429) and mouse UBF (aa 2–404) ref: https://doi.org/10.1016/j.molcel.2010.03.015. K562 cells were grown to subconfluence in Iscove's Modified Dulbecco's Medium, 10% fetal bovine serum. Immunoprecipitated chromatin was treated with RNaseA and the DNA isolated using 2% Na SDS and 2mg.ml-1 Proteinase-K. ChIP DNA samples were quality controlled by qPCR before being sent for library preparation and 50 base single-end sequencing on an Illumina HiSeq 2000 (McGill University and Genome Qubec Innovation Centre).