GSM7932557: S15; Homo sapiens; RNA-Seq
Patient tumors were graded pathologically by the study neuropathologists (CMH, NW) according to the World Health Organization classification [14]. At least 500 mg of the viable tumor was required to obtain enough for analysis and was processed within 30 min-1hour after resection. The normal brain was obtained from subjects as part of the planned surgical approach to gain access to a low-grade non-infiltrating glioma or during a planned super-total resection of adjacent regions that lacked T2/Flair abnormality on MRI imaging. The normal brain was sent for analysis separately from the tumor. The freshly resected tissue was processed in parallel both for a single cell suspension and formalin-fixed paraffin embedded (FFPE) analysis. For the former, the tissue was minced into small pieces using a scalpel, dissociated, and suspended using a Pasteur pipette in 10ml Iscove's DMEM (IMDM; Iscove's Modification of DMEM) 1X (Corning) containing 2% inactivated Fetal Bovine Serum (FBS; Sigma Aldrich) and Collagenase and DNase enzymes at final concentrations of 100μg/ml and 20units/ml, respectively. The prepared mixture was incubated for 35-40mins at 37°C with agitation. After tissue filtration using a 70-μm nylon cell strainer (BD Biosciences) and a brief centrifugation at 4°C, the pellet was resuspended in 20ml mix of 5.4ml of Percoll™ Plus (GE Healthcare) overlaid with 12ml of 1X Phosphate Buffered Saline (PBS) and 0.6ml of 10X PBS (Corning). The tube was centrifuged at 800gfor 10min at 4°C, with 9 acceleration and 0 deceleration. After centrifugation, the immune enriched cell pellet was collected, washed, stained with Trypan blue dye (Sigma-Aldrich), and counted using Countess II FL automated cell counter in a Countess cell counting chamber (Invitrogen). The FFPE slides were collected from the Neurological Surgery Tumor Bank (NSTB) of Northwestern University. Slides of tissue of 4 µm thickness were prepared, mounted on positively charged glass slides (Super Frost Plus microscope slides, ThermoFisher), and stored at room temperature for subsequent staining analysis. For each case, 1 H&E slide was reviewed, and tissue was segmented by a certified neuropathologist (CMH). Single-cell sequencing was carried out using the chromium Next GEM Single Cell protocol (10x genomics). Post-library preparation cells were sequenced using the Illumina Novaseq