GSM8759038: DLGG181; Homo sapiens; RNA-Seq
Snap frozen tissue samples from brain tumor resection specimens (5-15 mg) were thawed in 1 ml of HB buffer (0.25 m sucrose, 25 mm KCl, 5 mm MgCl2, 20 mm Tricine-KOH pH 7.8, 0.15 mm spermine tetrahydrochloride, 0.5 mm spermidine trihydrochloride, 1 mm DTT).  The tissue was then transferred to a 7-ml dounce homogenizer.  To this, 340 μl of 5% IGEPAL CA-630 (Sigma-Aldrich) and 4 ml of HB buffer were added. Tissue homogenization was performed using a tight pestle with 10-15 strokes. The homogenate was transferred to a 15-ml tube and the total volume was adjusted to 10 ml with 50% iodixanol solution.  A discontinuous iodixanol density gradient was prepared in a separate Corex tube (ThermoFisher) by layering 1 ml of 30% iodixanol solution over 1 ml of 40% iodixanol solution. The 9 ml tissue homogenate was carefully layered on top of the iodixanol cushion. Nuclei were isolated by centrifugation at 10,000 x g for 18 minutes.  The nuclei-enriched fraction at the 30-40% iodixanol interface (approximately 1 ml) was carefully collected. The concentration of nuclei in the isolated fraction was determined using a hemocytometer. The sample was then diluted to a final concentration of 100,000 nuclei/ml using 30% iodixanol solution supplemented with Protector RNase Inhibitor (Sigma-Aldrich). Single-nucleus encapsulation was performed using the 10x Genomics Chromium platform according to the manufacturer's instructions.  Libraries were generated using the 10x Genomics multiomics kit (version 2) and sequenced on the Illumina NovaSeq 6000 S4 platform at the SickKids Center for Applied Genomics (TCAG). Please note that while both gene expression (GEX) and assay for transposase accessible chromatin (ATAC) libraries were constructed, in the related manuscript only GEX data was used and therefore only that is submitted here.