title: WT-RUNX1
organism: Homo sapiens
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-05-13T17:05:14Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-07-24T10:36:59Z
attributes.tag: External Id
attributes.value: SAMEA5807730
attributes.tag: INSDC center name
attributes.value: TMU Research Center of Cancer Translational Medicine, Taipei Medical University
attributes.tag: INSDC first public
attributes.value: 2020-05-13T17:05:14Z
attributes.tag: INSDC last update
attributes.value: 2019-07-24T10:36:59Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8177:WT-RUNX1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 697
attributes.tag: cell type
attributes.value: pre-B cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: B-cell acute lymphoblastic leukemia
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: sample name
attributes.value: E-MTAB-8177:WT-RUNX1
attributes.tag: scientific_name
attributes.value: Homo sapiens