Illumina NovaSeq 6000 paired end sequencing; Cut&Run-seq of MUTZ3 cells to investigate changes in a translocated enhancer following CRISPR treatment
Cut&Run-seq of MUTZ3 cells to investigate changes in a translocated enhancer following CRISPR treatment
H3K27Ac Cut&Run libraries for the MUTZ3 bulk and sorted fragments were generated with an input of 200.000 cells.  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. The protocol described by the Henikoff group was used to generate these tracks (Skene et al., 2018), using a 0.04% Digitonin buffer and with the addition of cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche) and 1M Sodiumbutyrate (Sigma Aldrich) to all the buffers. Isolation was done according to the standard Phenol Chloroform protocol. Cut&Run samples were processed according to the protocol described by the Fazzio group