title: Illumina NovaSeq 6000 paired end sequencing; ChIP-seq of MUTZ3 cells in experiments to investigate EVI1 regulation by CRISPR or compound treatment
design: ChIP-seq of MUTZ3 cells in experiments to investigate EVI1 regulation by CRISPR or compound treatment
library_construction_protocol: 10 million cells were crosslinked by incubation with formaldehyde (final concentration 1%; Sigma-Aldrich) for 10 minutes, followed by quenching with glycine (final concentration 0.125 M; Sigma-Aldrich). Cells were pelleted, washed twice with ice-cold PBS and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF) and fresh protease inhibitor (Sigma) The MUTZ3 cell lines were cultured in αMEM (HyClone) with 20% fetal calf serum (FCS) and 20% conditioned 5637 medium. The 293T were cultured in DMEM (Gibco) with 10% FCS. All cell lines were supplemented with 50 U/mL penicillin and 50 μg/mL streptomycin. Viable frozen AML cells were thawed and resuspended in IMDM medium supplemented with: 20% BIT medium (Stem cell technologies), 1x β-mercaptoethanol (1000x Life technologies), 6 µg/ml LDL (Sigma Aldrich), human IL6, IL3, G-CSF, GM-CSF at 20 ng/ml and FLT3, SCF at 50 ng/ml (Peprotech. For measuring viability of MUTZ3 or primary AMLs, cells were seeded in an opaque colored 96-well plate at 15.000 cells/well in a total volume of 100 µl medium containing MYBMIM or control peptide TG3 at indicated concentrations (20 µM MYBMIM or control peptide TG3 for primary AMLs).  Material was sonicated to yield DNA fragments of ~300bp using a Bioruptor device (Diagenode). As input control 2%  of the chromatin was kept aside and the remainder was used for IP after addition of 5 volumes of IP dilution buffer (1.1% Triton X-100, 0.01% SDS, 167 mM NaCl, 16.7 mM TrisHCl pH 8.0, 1.2 mM EDTA) and fresh protease inhibitor (Sigma). ChIP samples were processed according to the Illumina TruSeq ChIP Sample Preparation Protocol (Illumina) or Diagenode Library V3 preparation protocol (Diagenode)