RNA-seq of medulloblastoma spheroid models

Studying medulloblastoma, the most common malignant paediatric brain tumour, requires simple yet realistic in vitro models. In this study, we optimised a robust, reliable, three-dimensional (3D) culture method for medulloblastoma able to recapitulate the spatial conformation, cell-cell and cellmatrix interactions that exist in vivo and in patient tumours. We investigated the initial stages of metastatic dissemination using brain-specific hyaluronan hydrogel matrices. In order to perform NGS of SHH medulloblastoma metastasis models, it was essential to extract high quality RNA for library preparation and sequencing. However, RNA isolation from hydrogels proved particularly difficult due to the low yields of RNA obtained. To efficiently utilise our samples, we chose to adopt two NGS technologies: traditional mRNA sequencing, which required at least 100 ng of RNA per sample, and 3' UPX sequencing, a method used for low input samples that sequenced the 3' end of RNA near the poly-A tail. Firstly, RNA was extracted from samples using the NucleoSpin RNA Plus kit (Macherey-Nagel). Following extraction, samples were shipped to QIAGEN Genomic Services (Hilden, Germany) where the subsequent steps of NGS were performed. Quality control of the RNA was performed using the Qubit RNA high sensitivity assay (Invitrogen) which quantified the amount of RNA in each sample. Samples with 100 ng or more of RNA in total were suitable for mRNA NGS. Samples with lower RNA yields were lyophilised and resuspended in a smaller volume of RNase-free water. Another quality control step was performed to quantify the RNA. Samples with an RNA concentration above 1.4 ng/mL were appropriate for 3' UPX NGS. However, samples with a lower RNA concentration were unsuitable for either method of NGS. 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. All prepared libraries successfully passed QIAGEN’s internal quality control checks and were sequenced on a NextSeq 500 Illumina sequencer. Following sequencing, FastQC analysis of the sequencing data was performed and all samples were considered suitable for downstream primary and secondary data analysis.