Targeted sequencing of PDX C0592-225
Flash frozen xenograft tumour samples were defrosted. Genomic DNA was extracted using the QIAAmp DNA Mini kit (Qiagen), following manufacturers protocol for extraction from tissues, with the exception that double the volume of reagents was used prior to loading onto DNA columns (buffers AL and ATL, proteinase K, ethanol). DNA yield was quantified using QUBIT fluorometer and dsDNA BR Assay Kit (Thermo Fisher Scientific). Aliquots containing 8-10 mg DNA were set aside for sequencing library preparation. Nextera adaptor sequences were appended to the 5' end of the sgRNA region primers with the addition of single nucleotides to allow downstream barcoded adaptor attachment by PCR. Multiplex PCR was performed using a T100 Thermal Cycler and Platinum Multiplex PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA). Enzymatic cleanup of the PCR product was carried out using ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific). Barcode PCR was completed using T100 Thermal Cycler, Nextera XT Index Kit v2 (Bio-Rad Laboratories), and FastStart High Fidelity PCR System (Sigma). The PCR products (approximately 400 bp, including adaptors) were size selected and cleaned using SPRIselect beads (Beckman Coulter) with a 1:1.2 library:beads ratio. The DNA size was analyzed using an High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA) on an 2100 Bioanalyzer (Agilent Technologies) and the DNA was quantified using dsDNA BR Assay Kit (Invitrogen, Carslbad, CA, USA) on a Qubit 4 Fluorometer (Invitrogen). Samples were normalized and pooled to create a library and each library was denatured to 2 nM or 4 nM. Sequencing runs pooling up to 12 sample libraries were carried out on a MiSeq benchtop sequencer (Illumina, San Diego, CA, USA) according to manufacturers protocol for 2 x 151 bp pair-end runs using MiSeq Reagent Kits v2 300 Cycle (Illumina). Sequencing runs pooling 13 - 47 samples libraries were carried out on a NextSeq sequencer (Illumina) according to manufacturers protocol for 2 x 151 bp pair-end runs using NextSeq 500 System Mid-Output Kit 300 Cycle (Illumina).
C0592-225