24_7_sgRNA1_GFPMin
Protocols: After a minimum of 72 hrs post nucleofection DNA or RNA was extracted (DNA Quick extract, Epicenter or Qiagen Allprep DNA/RNA mini, #80204) or cells were harvested for further analysis by respectively PCR, qPCR or flow cytometry analysis/FACS sorting. K562 cells were cultured in RPMI 1640 + L-glutamine (Hyclone SH30027.LS), 10% fetal calf serum (FCS, Gibco) and 50 U/mL penicillin and 50 μg/mL streptomycin (Gibco 15140-163). Cells were incubated at 37°C and 5% CO2 and passaged every 3-4 days to 100.000 cells/ml. CRISPR-Cas9 technology was used to make mutations or deletions in the regions described in the results section. Immunoprecipitation of crosslinked chromatin was performed with antibodies directed against H3K27Ac (Diagenode C15410196), H3K9Ac (Diagenode C15410004), H3K4me3 (Diagenode C15410003) or CTCF (Cell Signalling, 2899S). Crosslinks were reversed overnight at 65°C in the presence of proteinase K (New England Biolabs). De-crosslinked material was purified using a QIAGEN PCR Purification Kit. The purified DNA was processed according to the Nextflex ChIP Sample Preparation Protocol (Perkin Elmer) or the Microplex library preparation kit V2 (Diagnode C05010013) and sequenced on the Illumina NovaSeq6000 platform.
ENA first public
2021-07-26
ENA last update
2021-07-26
External Id
SAMEA7745867
INSDC center alias
Department of Hematology, Erasmus University Medical Center
INSDC center name
Department of Hematology, Erasmus University Medical Center
INSDC first public
2021-07-26T12:17:16Z
INSDC last update
2021-07-26T12:17:16Z
INSDC status
public
Submitter Id
E-MTAB-9965:24_7_sgRNA1_GFPMin
broker name
ArrayExpress
cell line
K562
cell type
myeloblast
common name
human
developmental stage
adult
disease
acute myeloid leukemia
genotype
t(3;8)
organism part
bone marrow
sample name
E-MTAB-9965:24_7_sgRNA1_GFPMin