GSM8421483: E1_hap_5_none_none_none_DHFR_cj_3; Homo sapiens; OTHER
gDNA was isolated using DNeasy columns (QIAGEN) according to the manufacturer's instructions, including a 10-min incubation at 56 °C during the proteinase K digestion step; DNA was eluted with 100 μl EB buffer. 20 cycles of primary PCR to amplify the region of interest was performed using ~200ng µL of genomic DNA in a 10 µL Platinum SuperFi II polymerase reaction (Thermo Fisher Scientific; for primers see Supplementary Data Set 1). Illumina adapters and indexing sequences were added via 15 cycles of secondary PCR with 1 µL of primary PCR product in a 10 µL Platinum SuperFi II polymerase reaction. The final amplicons were run on a TAE-agarose gel (0.7%); and the product band was excised and extracted using the Freeze and Squeeze Kit according to the manufacturer's instructions (Bio-Rad). Gel-purified amplicons were quantified with a dsDNA HS Assay kit on a Qubit fluorometer (ThermoFisher Scientific). Then, amplicons were pooled and sequenced on the NextSeq 550 platform (Nextseq 500/550 Mid Output Kit v2.5, Illumina, for primers see Supplementary Data Set 1). DHFR CRISPR-C time course samples in Fig. 3D were reported previously described, and subsequently sequenced for this study