NextSeq 500 sequencing; RNA-seq of medulloblastoma spheroid models
RNA-seq of medulloblastoma spheroid models
Ten wells per condition were harvested and pooled into a 1.5 mL microcentrifuge tube and snapfrozen in liquid nitrogen and stored at -80°C. RNA isolation was performed using the NucleoSpin RNA Plus kit (Macherey-Nagel, 740984). Samples were transferred to tubes containing lysing Matrix D beads (MP Biomedicals, 11412420), 1.4mm ceramic spheres used for grinding matrices. 350 µL of lysis buffer was added to each tube and samples were homogenised for 30 seconds using a FastPrep24 tissue homogeniser (MP  Biomedicals, 12079310). Samples were then transferred to a new 1.5 mL microcentrifuge tube and centrifuged at 11,000 x g for 5 minutes to pellet the hydrogel at the bottom of the tube. The rest of the extraction was performed according to the RNA isolation kit manufacturer's instructions. Following RNA extraction and quantification, libraries were prepared and sequenced. For mRNA NGS, library preparation was carried out using the TruSeq Stranded mRNA library preparation kit. All prepared libraries successfully passed QIAGEN's internal quality control checks and were subsequently sequenced on a NextSeq 500 Illumina sequencer. Following sequencing, quality control of the sequencing data was performed using FastQC analysis. All samples had high quality scores, indicating good technical performance of the sequencing. For 3' UPX NGS, library preparation was performed using the QIAseq UPX 3' Transcriptome kit.