Single-cell RNA-sequencing of peripheral blood in new-onset type 1 diabetes

Peripheral blood samples from newly diabetic T1D donors were collected in EDTA-coated tubes. Red blood cells were lysed using RBC (Red Blood Cell) Lysis buffer (Roche, 11814389001, Basel, Switzerland) according to manufacturer’s instructions. Briefly, 2 mL whole blood was resuspended in 4 mL of RBC Lysis buffer and incubated on shaker (700 rpm) for 15 min. After centrifugation (5 min at 500´g), the pellet was resuspended in 4 mL of RBC Lysis buffer and spun down again. Cells were resuspended in PBS with 1mM EDTA (Corning, 46-034-Cl, Corning, NY, USA). All procedures were conducted at RT (room temperature) and under sterile conditions. Isolated cells were then kept on ice before proceeding to the preparation for single-cell sequencing. Samples were counted on a LUNA-FL Automated Cell Counter (Logos Biosystems, South Korea) and were loaded on a BD Rhapsody (BD Biosciences, Franklin Lakes, NJ, USA) cartridge with a targeted capture of 25,000-40,000 cells. Reverse transcription, cDNA amplification and library construction were performed following the manufacturer’s instructions (BD Biosciences, Cat No. 23-24117(02)). Libraries were sequenced on a NovaSeq6000 or NextSeq2000 flow cell (Illumina, San Diego, CA, USA). The BD Rhapsody Sequence Analysis Pipeline (BD; version 1.12) was used to map the FASTQ files to the human reference genome (GRCh38).