GSM8794682: control_brain_1; Mus musculus; RNA-Seq
Once single-cells suspensions were generated for each sample condition, approximately 30,000 cells were loaded directly into a designated HIVE CLX collector (HIVE Collector, Honeycomb Biotechnologies, Inc. USA) in 1% FBS in DPBS. Single-cells settled into picowells of the CLX collector containing mRNA-capture beads. Media was removed from the collector and 2 mL of CLX sample wash solution was added. Then 1 mL of cell preservation solution was added. HIVE CLX collectors corresponding to each sample and group condition were stored at -80°C until transferred to the Australian Genome Research Facility Ltd. (AGRF, Melbourne, Australia) for single-cell NGS library processing and transcriptome recovery as per manufacturer's instructions. For transcriptome recovery, HIVE CLX collectors were sealed with a semi-permeable membrane, allowing for the addition of strong lysis solution and hybridization solution. Capture beads with transcripts were extracted from the collector by centrifugation. Remaining library preparation steps were performed in a 96-well format. Library size and assessment was performed using TapeStation software and the High Sensitivity D5000 ScreenTape assay (Agilent Technologies, CA, USA). Final pooled libraries were determined by qPCR. HIVE scRNA-sequencing libraries were sequenced using primers contained in the kit