10x Visium spatial RNA-Seq of human GBM cell-line derived murine orthotopic xenograft
The brain was removed and dissected into 2 mm coronal slabs. Fresh tissues were embedded in Tissue Tek OCT compound (Fisher Scientific 14-373-65) and snap-frozen in a chilled isopentane and dry ice bath. 10 m cryosections were mounted on barcoded Visium slides (10x Genomics), and libraries prepared with the 10x Visium Spatial Gene Expression kit per manufacturer's protocol. Briefly, sections were fixed in methanol, stained with H&E, and scanned on an EVOS FL Auto Imaging System (Thermo Scientific) using a 10x objective. Permeabilization, reverse transcription, second strand synthesis, denaturation and cDNA synthesis were performed as per protocol. Cycle number determination for cDNA amplification was performed on a BioRad Realtime qPCR system using KAPA SYBR FAST qPCR Master Mix (Roche, KK4600). cDNA QC and qualification was performed on an Agilent 2100 Bioanalyzer with Agilent High sensitivity DNA chips (Agilent 5067-4626). After enzymatic fragmentation and double size selection using SPRIselect reagent (Beckman Coulter, B23318), unique indexes and P5 and P7 Illumina primers were added to the libraries using Dual Index Kit TT Set A (PN-1000215). Libraries were sequenced on an Illumina NextSeq500/550 instrument using paired-end sequencing with the following parameters: 28 cycles for Read1 and 90 cycles for Read ,10-10 cycles for index i7 and index i5, loading concentration 1.8pM on NextSeq 500/550 High Output Kit v2.5 150cycle (Illumina, 20024904).