Ambion rnase a

The reference material of the investigational CAR-T construct was prepared in-house (4.3 μg/μL) with a proprietary target binder. Primers and TaqMan probes used for droplet digital PCR assay were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). The yeast HIS3 and LEU2 plasmid DNA were obtained from Aldevron, LLC (Fargo, ND). Bio-Rad QX200 AutoDG Droplet Digital PCR System™ including automated droplet generator, C1000 Touch™ Thermal Cycler, and QX200 droplet reader and their consumables were from Bio-Rad Laboratories (Hercules, CA). DNeasy Blood & Tissue Kit is from Qiagen (Valencia, CA). KingFisher magnetic beads processor™, MagMAX™ DNA Multi-Sample Ultra 2.0 Kit, and Ambion™ RNase A were from Thermo Fisher Scientific (Waltham, MA). GentleMACS Octo Dissociator was from Miltenyi Biotec (Somerville, MA).

gDNA extraction from mouse blood and tissue homogenate was conducted with MagMAX™ DNA Multi-Sample Ultra 2.0 Kit with minor modifications. The gDNA was also extracted from mouse blood and tissue samples using MagMAX™ DNA Multi-Sample Ultra 2.0 Kit in the presence of Proteinase K. Ambion™ RNase A (5 μL each, affinity purified, Thermo Fisher Scientific) was added to make sure the contamination of RNA is negligible. Tissue samples were homogenized in nine volumes of PBS containing 15 mM EDTA with gentleMACS Octo Dissociator in the mode of RNA_01_01. Binding enhancer solution (10 or 40 μL), homogenate solution (100 or 400 μL), and proteinase K (10 or 40 μL) were mixed well in a plate and then incubated the plate at 65 °C overnight. The DNA extraction process was automated with the KingFisher™ Flex system using the sample processing programs (MagMAX_Ultra2_200μL_Flex and MagMAX_Ultra 2_Tissue_V) according to the manufacture’s protocol. The gDNA purity from each extracted sample was monitored by NanoDrop 2000 spectrophotometer using the absorbance ratio at 260 and 280 nm (A₂₆₀/A₂₈₀).