Integration site characterization of clone 31/NISTCHO, a CHO cell line expressing a non-originator version of NISTmAb
Samples of Clone 31 research cell bank (RCB) cultures were collected within 2 passages of thaw and were prepared using the DNeasy Blood & Tissue Kit (Qiagen, cat. 69504) using a QIACube (Qiagen) automated DNA extraction platform according to the manufacturers protocol. Purified DNA was quantified via Nanodrop (ThermoFisher) or using a Qubit fluorometer (ThermoFisher). For gDNA cleavage, library construction, and sequencing, 30 L of the pre-assembled RGEN RNP was added to 20 L of genomic DNA (gDNA; 10 g) and incubated at 37C for 1 h. The reaction was stopped by heating it to 65C for 10 min. The terminated RGEN reactions were treated with RNase cocktail (Thermo Fisher Scientific) and Proteinase K (Qiagen), followed by purification of cleaved gDNA with Beckman SPRIselect paramagnetic beads according to the manufacturer's instructions. Purified cleaved gDNA was end-repaired and A-tailed with Klenow Fragment (3',5' exo-) from NEB, followed by ligating DNA sequencing adapters in ligation buffer from the Oxford Nanopore Ligation Sequencing Kit (ONT, LSK109) using Quick Ligase (NEB) for 10 min at room temperature. Samples were sequenced using a R9.4.1 flow cell on a GridION sequencer following the manufacturer's protocol for ligation-based sequencing of gDNA using the MinKNOW software (v. 19.2.2). Base calling was performed using GUPPY (v.3.0.3, Oxford Nanopore) to generate FASTQ sequencing reads from the electrical data. Reads were aligned to the custom reference genome containing the Chinese hamster reference genome (CriGri-PICRH-1.012) and cNISTmAb expression vector sequence using minimap228 (v.2.17). Programs such as Burrows-Wheeler Aligner (BWA MEM29), samtools30,31, bbmap package32, IGV33,34, and Phrap/Consed35 (v. 29) were used for extracting and visualizing sequence context of vector integration sites.