Mitogenic and progenitor programs in single pilocytic astrocytoma cells.

Zachary J. Reitman*, Brenton R. Paolella*, Guillaume Bergthold, Kristine Pelton, Sarah Becker, Robert Jones, Claire E. Sinai, Hayley Malkin, Ying Huang, Leslie Grimmet, Zachary T. Herbert, Yu Sun, Jessica L. Weatherbee, John Alberta, John F. Daley, Orit Rozenblatt-Rosen, Alexandra L. Condurat, Kenin Qian, Prasidda Khadka, Rosalind A. Segal, Daphne Haas-Kogan, Mariella G. Filbin, Mario L. Suva, Aviv Regev, Charles Stiles, Mark W. Kieran, Liliana Goumnerova, Keith L. Ligon, Alex K. Shalek, Pratiti Bandopadhayay†#, Rameen Beroukhim†#

Contact: rameen_beroukhim@dfci.harvard.edu, pratiti_bandopadhayay@dfci.harvard.edu

Pilocytic astrocytomas are pediatric low grade gliomas that frequently contain oncogenic BRAF alterations.  To explore the effect of expressing oncogenic BRAF in brain progenitor cells, we examined transcriptomes of single mouse neural stem cells (mNSCs) that we engineered to express the KIAA1549-BRAF fusion that is frequently found in pilocytic astrocytomas.  In parallel, we examined mNSCs expressing BRAF-V600E, another common BRAF alteration found in pediatric gliomas, or vector only control.  Single cell RNA-seq data were generated for 487 mNSCs after quality filtering (n = 170 vector control mNSCs, n = 154 KIAA1549-BRAF mNSCs, and n = 163 BRAF-V600E mNSCs).  These mNSC data complement a human pilocytic astrocytoma dataset that was reported in the same study.

Workflow. Primary neural stem cell cultures were established from the medial ganglionic eminence of embryonic day 14 murine embryos and transduced with KIAA1549:BRAF15, BRAF-V600E, or empty vector retrovirus constructs.  Cells were dissociated to single-cell suspensions and sorted into 96-well plates. Full-length scRNA-seq was performed following the Smart-Seq2 protocol.