Dneasy blood and tissue kit

For RHAG gene sequence analysis, DNA was extracted from donor PBMCs or hiPSCs by an automated method using the QIAsymphony instrument (Qiagen, Germany). Primers to amplify RHAG gene exon 6 are listed in Table S2. For CRISPR edit validation, genomic DNA was isolated from each expanded clone using DNeasy Blood and Tissue Kit (Promega). DNA regions encompassing guide sites were amplified using specific primers (Table S2). Amplification was performed with SequalPrep™ Long PCR Kit with dNTPs (Applied Biosystems, USA). The PCR products were Sanger sequenced using the Big Dye Terminator v1.1 kit (Applied Biosystems). Sequencing primers are also listed in Table S2. DNA sequences were aligned with the reference genomic sequences: NG_011704.1 for the RHAG gene and NG_006669.2 for ABO gene, using the CLC GenomicWorkbench 21.0.3 software (Qiagen).

Diagnostic biopsy specimens submitted to our institution were routinely examined in the course of intraoperative consultations (frozen sections), followed by standard formalin-fixation and paraffin-embedding (FFPE) for histological workup in most cases. External cases were either received as FFPE blocks or as near-native tumour tissue submitted in SurePath^® (Becton Dickinson, USA) or ThinPrep^® preservatives (Hologic Inc., USA). For cryopreserved biopsies, one to four 70 μm thick cryosections were collected. For FFPE blocks, a series of 8 or 14 serial sections (4 μm) were collected on glass slides, and an additional H&E section in the middle of each series was used to verify tumour cell content. In samples containing significant non-neoplastic or necrotic areas on the cut surface, viable tumour cell-rich areas were manually microdissected. For native specimens, DNA was extracted with automated commercial systems: Promega Maxwell^® FFPE DNA extraction, Promega Maxwell^® blood DNA extraction, and Qiagen DNeasy^® Blood and Tissue kits on Maxwell or QiaCube^® instruments (Promega, USA; Qiagen, Germany). DNA from FFPE specimens was extracted with the Promega Maxwell^® FFPE DNA extraction, Qiagen DNeasy^®, and RecoverAll^® kits. Except for differences in average read lengths in nanopore sequencing runs for native materials, all of the above-mentioned kits produced technically valid results.