title: Illumina MiSeq paired end sequencing; Amplicon sequencing of a K562 cell line model with a patient-based t(3;8) translocation following CRISPR treatment
design: Amplicon sequencing of a K562 cell line model with a patient-based t(3;8) translocation following CRISPR treatment
library_construction_protocol: After a minimum of 72 hrs post nucleofection DNA or RNA was extracted (DNA Quick extract, Epicenter or Qiagen Allprep DNA/RNA mini, #80204) or cells were harvested for further analysis by respectively PCR, qPCR or flow cytometry analysis/FACS sorting.  K562 cells were cultured in RPMI 1640 + L-glutamine (Hyclone SH30027.LS), 10% fetal calf serum (FCS, Gibco) and 50 U/mL penicillin and 50 μg/mL streptomycin (Gibco 15140-163). Cells were incubated at 37°C and 5% CO2 and passaged every 3-4 days to 100.000 cells/ml. CRISPR-Cas9 technology was used to make mutations or deletions in the regions described in the results section.  After a minimum of 72 hrs post nucleofection DNA or RNA was extracted (DNA Quick extract, Epicenter or Qiagen Allprep DNA/RNA mini, #80204) or cells were harvested for further analysis by respectively PCR, qPCR or flow cytometry analysis/FACS sorting.  The first PCR was performed using Q5 polymerase (NEB), the second nested PCR with KAPA HiFi HotStart Ready mix (Roche).