GSM5958227: REVeRT (split) Prime Editor 2; Homo sapiens; OTHER
Transfected cells were detached by scraping them off into remaining culture medium. The cells suspension was centrifuged (10 min, 5000 x g, 4 °C) and the cell pellet resuspended in a cell lysis buffer (RLT buffer, Qiagen) supplemented with 1% β-mercaptoethanol. Cells were then disrupted using a mixer mill at 30 Hz for 1 min. The lysates were centrifuged (10 min,500 x g, 4 °C) and supernatant was added to gDNA extraction columns. The columns were centrifuged for 4 min at 1500 x g and 1 min at 10000 x g at RT and incubated with 400 µl of RNase wash (Genomic lysis buffer (Zymo Research) supplemented with RNase A (Sigma-Aldrich), 1:5000) for 10 min followed by a 2 min centrifugation at RT. Next, 400 µl of DNA Pre-Wash buffer (Zymo Research) and 600 µl of g-DNA Wash buffer (Zymo Research) were added and with intermediate centrifugation steps for 1 min at 10000 x g. 600 µl of g-DNA Wash buffer was added again and centrifuged for 2 min at 10000 x g. To elute gDNA, 100 µl of H2O were added to the column, incubated for 20 min at RT and centrifuged for 2 min at 10000 x g. gDNA concentrations und purity were measured using the NanodropTM 2000c spectrophotometer. For amplification of the respective target locus, 100 ng of the extracted gDNA were used in a PCR reaction with the Q5 high-fidelity DNA polymerase (New England Biolabs) according to the manufacturer's instructions. The PCR was performed using primers containing Illumina partial adapters. The PCR product was validated via gel electrophoresis. Subsequently, the PCR-amplified DNA was purified from the agarose gel with the QIAquick gel extraction kit (Qiagen) according to the manufacturer's instructions. The purified PCR product was sent to next-generation sequencing (GENEWIZ, Azenta Life Sciences).