P21_Tumor_sccloning_8
Bulk tumour spheres were successfully constructed using a previously reported method for tumour sphere isolation from the human brain31. Out of five tumours of P01, we selected T5 (posterior tumour) because of its highest viability. Within hours after the operation, meningioma-derived tumour spheres (TS) were generated from the patient's tumour tissue. The meningioma TSs were maintained in complete media consisting of Dulbeccos Modified Eagles Medium/F12 (Mediatech), 1x B27 (Invitrogen), 20 ng/mL basic fibroblast growth factor, and 20 ng/mL epidermal growth factor (Sigma-Aldrich). To establish separate clonal lines, meningioma TS were dissociated into single cells using Accutase. The cells were then diluted at a ratio of 1:100 and plated into 96-well plates. After 3 days, wells containing single cells were manually selected under a microscope, and the cells were gradually expanded.  Following the quality and quantity check using the cell counter, we extracted genomic DNA from each clone with the QIAamp micro-DNA kit (Qiagen) following the manufacturers instructions. For 8 clones of P01,  we established an exome library without further genomic amplification. For 8 clones derived from P21, we exploited primary template-directed amplification (PTA), due to the lack of the DNA amount to execute conventional WES. PTA was performed using the ResolveDNA Whole Genome Amplification Kit (BioSkryb PN100136, NC, USA) following the manufacturer's protocol. All PTA reagents were added step-by-step according to the provided instructions. The lysed samples, along with the enzymes and terminators, were incubated on a thermal cycler at 30C for 10 hours. This was followed by enzyme deactivation at 65C for 3 minutes. Finally, bead purification of the amplicons was conducted as instructed by the manufacturer. Then, ~300x exome libraries (Agilent SureSelect XT HS) were prepared for all clones of two individuals. The method of exome capture and sequencing is identical to the aforementioned. FASTQ files were processed and read alignment to GRCh38 was conducted accordingly.