title: GSM5848120: SS-5-86; Homo sapiens; OTHER
library_construction_protocol: Once tumor engraftment was confirmed using MRI, mice were randomly assigned to control or treatment groups and chemoradiotherapy treatment was started. For mice in the combined chemoradiotherapy arm, 50 mg/kg TMZ was oral gavaged one hour before cranial irradiation was administered to mice. The mouse shield is designed such that only the head of the mice is irradiated. Gentra Puregene Tissue kit was used to extract all genomic DNA from full mouse brains. Considering that the GBM tumor population in a mouse brain is expected to be significantly smaller, especially at the MRD timepoint, we optimized genomic DNA extraction protocols for snap-frozen mouse brain samples and performed nested-PCR to amplify the 137 bp barcode region for genomic sequencing. Spike-in control barcoded HEK293 cells were added to mouse brain samples for gDNA extraction to estimate clone size. We used NEB UltraQ II polymerase to amplify the 137 bp  barcode regions using nested-PCR, which were then visualized on a 3% agarose gel. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer's protocols.