GSM8879032: Patient_4_pre; Homo sapiens; RNA-Seq
venous blood samples were collected for testing in PAXgene™ blood RNA tubes (Qiagen, Hilden, Germany) and Acid Citric Dextrose tubes (BD).  Red blood cells were depleted from whole blood by immunomagnetic negative selection, using the EasySep™ RBC depletion reagent protocol (catalog #18000), preserving all leukocyte populations, including granulocytes. HIVE devices were each loaded with cells in 1 mL of DPBS + 1% FBS, followed by 3mL of cell media. The leukocytes were loaded into the HIVE system within an hour of blood sampling from all individuals to minimise neutrophil activation. Single-cells settled into picowells of the HIVE containing 3' transcript-capture beads. HIVEs were then placed onto a spin plate and spun at 30 x G for 3 minutes. Media was removed from the HIVE, and 2mL sample wash solution (provided by PerkinElmer) was added to each. Sample wash solution was removed, and the cell-loaded HIVE™ were frozen at -80°C after the addition of cell preservation solution. Following standard protocol, cell-loaded HIVE devices were sealed with a semi-permeable membrane, allowing for the use of the strong lysis solution followed by the addition of hybridization solution. After collection with or without storage, beads with captured transcripts were extracted from the HIVE device by centrifugation. The remaining HIVE library preparation steps were conducted in a 96-well plate format. PerkinElmer's LabChip® GX TouchTM nucleic acid analyzer was used for accurate assessment of individual library sizes, and the concentration of final pooled libraries was determined by qPCR. HIVE scRNAseq libraries were sequenced using specific primers contained in the kit on an Illumina® NovaSeq® 6000 sequencer