GSM8195237: 1_4; Homo sapiens; RNA-Seq
Human GBM fresh tumor tissue specimens were allocated by the Neuropathology Division and the Tissue and Blood Repository at Brigham and Women's Hospital in accordance with prospective consent to a tissue procurement IRB protocol for CNS disorders or IRB waiver of consent. Tissue was immediately placed in Hibernate A (Gibco) media using aseptic technique. Fragments of tissue were gently cut into ~2mm3 pieces for cultures on Millicell transwell membranes (Millipore Sigma) in neurobasal A media supplanted with B27 (Gibco). The transwell system contained 1mL media below the membrane, fitted into a 6 well tissue culture plate, with the slice resting on the membrane, covered with a thin film of media (roughly 5 mL) for optimal oxygen diffusion. The liquid film was maintained by pipetting 5 mL media onto slices from the lower chamber daily. Media in the lower well was replaced every 48 hours. The EF1a promoter was cloned into the pBOB-SynP-HTB plasmid, which contains a lentiviral backbone and was a gift from Edward Callaway & Liqun Luo (Addgene plasmid # 30195; http://n2t.net/addgene:30195 ; RRID:Addgene_30195). Lentivirus was produced from newly cloned pBOB-EF1a-HTB using previously described methods. On the first day of culture, each piece of tissue was lentivirally transduced with TVA and G under the EF1a promoter, which was pipetted on top of cultures in a volume 5 mL at a concentration of 106 infectious units. One day later, tissue was gently rinsed with PBS, which was removed to leave a thin liquid film on the top of each slice. After five days, tissues were injected with 1 mL barcoded, pseudotyped rabies virus (EnvA-RABDG) at a concentration of 10,000 infectious units per mL. One day later, tissue was gently rinsed with PBS, which was removed to leave a thin liquid film on the top of each slice. After five days, tissue was digested using papain, followed by trituration using fire polished glass Pasteur pipettes. Cells then underwent myelin removal using the Myelin removal kit from Miltenyi. Filtered cell suspensions then underwent FACS sorting for mCherry+ cells. Sorted mCherry+ cells then underwent single cell droplet encapsulation using Indrop v3 switch drops, followed by cDNA library preparation at the Single Cell Core at Harvard Medical School. Prior to sequencing, mCherry barcodes were amplified using 3'cDNA primers in addition to a primer to mCherry as described previously. Cells then underwent myelin removal using the Myelin removal kit from Miltenyi. Filtered cell suspensions then underwent FACS sorting for mCherry+ cells. Sorted mCherry+ cells then underwent single cell droplet encapsulation using Indrop v3 switch drops, followed by cDNA library preparation at the Single Cell Core at Harvard Medical School. Prior to sequencing, mCherry barcodes were amplified using 3'cDNA primers in addition to a primer to mCherry as described previously.