title: Longitudinal Single-Cell Transcriptomic Profiling of the Peripheral Immune Response in Ischaemic Stroke
design: Longitudinal Single-Cell Transcriptomic Profiling of the Peripheral Immune Response in Ischaemic Stroke
library_construction_protocol: Venous blood was drawn from ischaemic stroke patients within 96 h of stroke symptom onset or time last seen well, as well as at the 6-9-month follow-up timepoint. Blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes and processed within 4 h.   To obtain peripheral blood mononuclear cells (PBMCs), EDTA blood was diluted 1:1 with phosphate-buffered saline (PBS) supplemented with 2% foetal bovine serum (FBS) and centrifuged at 1200 g for 15 min at 20°C in SepMate™ PBMC Isolation Tubes (StemCell Technologies, Vancouver, Canada) with Ficoll-Paque™ PLUS density gradient media (Cat. No. 17144003; Cytiva Life Sciences, Amersham, UK). Following centrifugation, the top supernatant layer containing PBMCs was transferred to 50 mL falcon tubes, diluted with 20 mL PBS + 2% FBS and centrifuged at 500 g for 10 min at 20°C. Supernatant was discarded, and the cell pellet was resuspended in ACK Lysing Buffer (Cat. No. A1049201; Gibco, Waltham, USA) for 5 min at room temperature. PBS (10 mL) + 2% FBS was added and tubes centrifuged at 500 g for 5 min at 20°C. The cell pellet was resuspended in 20 mL FBS + 2% FBS and cells were counted before centrifugation at 500 g for 10 min at 20°C. Cells were resuspended at a final density of 5-6 × 106 cells / 500 µL CryoStor® CS10 Freezing Medium (Cat. No. 100-1061; StemCell Technologies), and frozen at -80°C in CoolCell™ Cell Freezing Vial Containers (Cat. No. 15542771; Corning, New York, USA). Finally, PBMC aliquots were transferred to liquid nitrogen until ready for use.  Approximately 33,000 cells per pool (8250 per individual, per stage) were loaded onto the 10x Genomics Chromium Controller. Cell lysis and RNA release occurred immediately after encapsulation within Gel Beads-in-emulsion (GEMs). Reverse transcription was performed inside the GEMs to generate barcoded cDNA using the Chromium Next GEM Single Cell 5' Reagent Kits (10x Genomics), according to the manufacturer's protocol. Following reverse transcription, the emulsion was broken and barcoded cDNA was purified and amplified. The cDNA was then fragmented, end-repaired, and A-tailed. 5' Gene Expression libraries were constructed by adaptor ligation and sample index PCR using the Chromium Next GEM Single Cell 5' Reagent Kits (10x Genomics). The resulting libraries were size-selected and purified.