title: TEM-GBM trial - scRNAseq and TCR
design: TEM-GBM trial - scRNAseq and TCR
library_construction_protocol: Fresh tumor material was obtained from some Temferon patients or other control patients treated according to standard-of-care when undergoing second surgery for progressive disease. The control patients have agreed to the use of their biological material for research purposes and signed the relevant informed consent forms from the Department of Neurosurgery of the San Raffaele Hospital. Tumor was dissociated using the ”Tumor Dissociation Kit” (Miltenyi Biotec), lysed to eliminate red blood cells with Ammonium Chloride Solution Lysis Buffer (ACK) (Stem Cell Technologies), purified using “Debris removal kit” (Miltenyi Biotec) and “Dead cell removal kit” (Miltenyi Biotec), and separated into a CD45+ and CD45- fraction by human CD45 (TIL) MicroBeads (Miltenyi Biotec). Both fractions were used, either fresh or frozen, for 10x single-cell RNA sequencing. Single cell RNA and V(D)J libraries were prepared with the Chromium Next GEM Single Cell 5' and V(D)J Reagents Kits v1.1 (10X Genomics). Briefly, after CD45 selection, at least 900 cells were resuspended at the appropriate concentration for loading into the Chromium Next GEM Single Cell 5' and V(D)J Reagents Kits v1.1chip according to the manufacturer's protocol