title: Illumina MiSeq paired end sequencing; Amplicon-seq of an enhancer region targeted by CRISPR in MUTZ3 to assess the mutations introduced by different sgRNA guides
design: Amplicon-seq of an enhancer region targeted by CRISPR in MUTZ3 to assess the mutations introduced by different sgRNA guides
library_construction_protocol: RNP complexes were formed incubating sgRNA and Cas9 (IDT) for 20-30 at RT before nucleofection using the Neon (Thermofischer) with buffer R using program 3.  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. Genomic DNA was extracted 48 or 72 h after transfection using Quick Extract buffer (Epicenter) For amplicon sequencing we used the Illumina PCR-based custom amplicon sequencing method using the TruSeq Custom Amplicon index kit (Illumina). The first PCR was performed using Q5 polymerase (NEB), the second nested PCR with KAPA HiFi HotStart Ready mix (KapaBiosystems).