GSM8864451: Patient A oligodendroglioma tumor resection; Homo sapiens; RNA-Seq
Tumor resection tissue was frozen on dry ice and stored at -80°C until nuclei isolation. All steps involved in isolation of nuclei were conducted using pre-chilled buffers and a centrifuge pre-set to 4°C. Tissue was transferred to a Dounce homogeniser containing homogenisation buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris pH 8.0, 1 mM dithiothreitol (DTT), 0.2 U/μl RNasin ribonuclease inhibitor, and 0.1% Triton X-100) and incubated for one minute before homogenization. Tissue was homogenized using a combination of loose and/or tight pestles and a 1 ml pipette tip, depending on the individual piece of tissue. The resulting homogenates were filtered through a 30 μm strainer and centrifuged at 500 × g for 5 minutes. Supernatant was discarded and the pellet was resuspended in wash and resuspension buffer (Dulbecco's PBS containing 1% bovine serum albumin (BSA), 1 mM DTT, and 0.2 U/μl RNasin ribonuclease inhibitor). A further 1-2 washes were conducted, depending on pellet size. Nuclei were counted using trypan blue and a haemocytometer. Nuclei were then fixed using the Chromium Next GEM single cell fixed RNA sample preparation kit (10x Genomics) and stored at -80°C according to the manufacturer's instructions. Fixed nuclei were barcoded using the human transcriptome Chromium Fixed RNA kit, human transcriptome (4 reactions x 4-3 barcodes) (10x Genomics) with probe hybridisation conducted according to the manufacturer's instructions. Nuclei were targeted on the Chromium controller, after which library preparation was conducted. Library preparation was performed using the Chromium Single Cell Gene Expression Flex Kit (10X Genomics) according to the manufacturer's instructions. Briefly, single nuclei were encapsulated into Gel Bead-In-Emulsions (GEMs), where poly-A RNA was reverse transcribed to cDNA incorporating a Unique Molecular Identifier (UMI) and a 10X Barcode. Adapter ligation was performed to generate paired-end libraries with P5 and P7 adapters and multiplexed for Illumina sequencing. Final libraries were quality checked and sequenced on an Illumina platform.