GSE111734
Astrocytic trans-differentiation completes a multicellular paracrine feedback loop required for medulloblastoma tumor growth
We used RNA-sequencing technology for high-throughput profiling of tumor-derived astrocytes. Laser capture microdissection of individual cells was performed on an ArcturusXT LCM system (Applied Biosystems) with an infrared laser using CapSure HS LCM caps after optimization of laser power and duration. For each sample, 250 cells were microdissected per LCM cap and split into five 50-cell technical replicates (as a control, one replicate did not undergo reverse transcription) after elution from the cap. Reamplified cDNA (~500 bp 3' ends) was purified away from primer concatemers by two rounds of purification with 0.7x volume of AMPure XP beads (Beckman) according to the manufacturer's recommendations, and purified cDNA was quantified by Qubit assay (Life Technologies). For each sample, 1 ng was tagmented with the Nextera XT kit (Illumina) and sequenced as 75 bp paired-end reads on a NextSeq instrument using v2 reagents (Illumina).  14–22 million reads per sample were filtered for signal to noise (chastity filtered), assessed for overall quality with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and then mapped with STAR (Dobin et al., 2013) against the mouse genome build mm10. Overall design: Comparing the gene profile of tumor derived astrocytes with normal astrocytes and tumor cells (TuGNPs)