GSM7768012: AD patient, PBMC SNUBH-38-2; Homo sapiens; OTHER
All peripheral blood samples were collected by EDTA tubes (BD Vacutainer K2EDTA tubes, 367525) at least 15 ml in total per a patient29. Peripheral blood mononuclear cells (PBMCs) and plasma were separated by density-gradient centrifugation using Ficoll (Cytiva Ficoll-Paque Plus, 17-1440-02) solution. After the PBMC suspension was resuspended in CELLBANKER (Amsbio CELLBANKER 2, 11914), the collected PBMCs were immediately cryopreserved and stored in a Freezing container (Nalgene Mr. Frosty, C1562) for a week. Total RNA was isolated by TRIzol Reagent (Invitrogen TRIzol Reagents, 15596026) following the manufacturer's instructions. For the NGS library preparation of BCR heavy chain sequence, genes encoding the variable region of the heavy chain (VH) at the 5 prime forward part and the first constant region of the heavy chain (CH1) domain at the 3 prime reverse part were amplified by specific PCR primers as reported previously16. All primer sequences are listed in Extended Table 2. In short, one microgram of total RNA was used as the input template for library preparation. Next, reverse transcription was conducted as following the manufacturer's instructions using SuperScript IV First-Strand Synthesis System (Invitrogen, 18091050) with CH1 gene-specific primers for five immunoglobulin heavy chain isotypes containing UMI (unique molecular identifiers) barcodes that consist of 12 random nucleotides, 14 base pair in total; NNNNTNNNNTNNNN, and partial Illumina adapters (No. 1-8). After first-strand cDNA synthesis, it was purified with SPRI beads (Beckman Coulter AMPure XP Reagents, A63882) at a 1:1.8 ratio and second-strand cDNA was synthesized using a KAPA Biosystems kit (Roche KAPA HiFi Hotstart PCR kit, KK2502) with VH gene-specific primers (No. 9-14; 95°C for 3 min, 98°C for 30 s, 60°C for 45 s, and 72°C for 6 min). Double-stranded cDNA was purified through SPRI beads at a 1:1 ratio. Following that, it was amplified using KAPA Biosystems kit and double primers containing Illumina adapters and index sequences (No. 15, 16; 95°C for 3 min, 25 cycles of 98°C for 30 s, 60°C for 30 s, 72°C for 1 min, and 72°C for 5 min). The PCR products were subjected to electrophoresis on a 1.5% agarose gel to separate through the length of DNA and purified using QIAquick gel extraction kit (QIAGEN, 28704) following the manufacturer's protocols. The final NGS libraries were obtained using SPRI beads at a 1:1 ratio and quantified with a 4200 TapeStation System (Agilent Technologies, G2991BA) for QC. Libraries with a single peak of appropriate length were subjected to NGS analysis using the Illumina Nocaseq6000 platform.