KO-RUNX1
Homo sapiens
Protocols: CRISPR/Cas9 mediated KO of MED1: pSpCas9(BB)-2A-GFP (PX458) was purchased from Addgene. Guided RNA (gRNA) targeting exon 5 (5'-GAAGTGCAGTTAGATCCTGCAGG-3') or exon 13 (5'-CCAACTTATGCACCCCTGTATGA-3') of MED1 was constructed into the vector. 697 cells were transient transfected by Lipofectamine 3000 (Invitrogen) and GFP+ cells were sorted on day 3 for expansion as single clones. Isolation, subcloning and sequencing of edited genomic DNA was performed from samples on day 28. Expression of MED1 was confirmed by immunoblot with anti-MED1 antibody (Bethyl laboratories, A300-793A). 697 cells were cultured in RPMI 1640 (Invitrogen) and 10% FBS. Immunoprecipitation was performed according to the manufacturer's protocol (Upstate Biotechnology, Inc., Lake Placid, NY) with slight modifications. 697 cells were fixed for 10 min with 1% of formaldehyde at room temperature, and then quenched with a final concentration of 1M of glycine. The cells were lysed and the chromatin was sonicated to 200-500 bp fragments with a Bioruptor (Diagenode, Sparta, NJ). Chromatin was immunoprecipitated by the anti-MED1 (Bethyl laboratories, A300-793A) and anti-RUNX1 (Abcam, ab23980) antibody with Protein G Dynabeads (Invitrogen). The beads were washed once with each washing buffer, including low salt immune complex wash buffer, high salt immune complex wash buffer, and LiCl immune complex wash buffer, and twice with 1X TE buffer. Precipitates were eluted with 1% of SDS and 100 mM of NaHCO3. The samples were heated at 65°C for 6 hr in order to reverse cross-link, and purified with ChIP DNA Clean & Concentrator (Zymo Research, D5201). The sequencing libraries were constructed from 20 ng of immunoprecipitated and input DNA. End pairing (End-It, ER81050, Epicentre), 3'-end A-tailing (Klenow, NEB), and barcode ligation (NEXTflex ChIP-seq Barcode-12, 514121, Perkin Elmer) was performed according to manufacturer's instruction. Barcoded DNA was purified by Agencourt AMPure XP (A63880, Beckman) and amplified by PCR (Phusion HF, F530S, ThermoFisher Scientific) for 16-cycles.
ENA-FIRST-PUBLIC
2020-05-13T17:05:14Z
ENA-LAST-UPDATE
2019-07-24T10:36:59Z
External Id
SAMEA5807727
INSDC center name
TMU Research Center of Cancer Translational Medicine, Taipei Medical University
INSDC first public
2020-05-13T17:05:14Z
INSDC last update
2019-07-24T10:36:59Z
INSDC status
public
Submitter Id
E-MTAB-8177:KO-RUNX1
broker name
ArrayExpress
cell line
697
cell type
pre-B cell
common name
human
disease
B-cell acute lymphoblastic leukemia
genotype
CRISPR/Cas9 mediated knockout of MED1
organism part
bone marrow
sample name
E-MTAB-8177:KO-RUNX1
scientific_name
Homo sapiens