title: GSM8128159: Mouse Notch1-WT, IDHi treated, Chromium gene expression; Mus musculus; RNA-Seq
library_construction_protocol: Fresh specimens were mechanically dissociated with a disposable, sterile scalpel and further enzymatically dissociated into single cell suspensions using the enzymatic brain dissociation kit (papain-based) from Miltenyi Biotec as previously reported. Tumor cells were blocked in 1% bovine serum albumin in phosphate-buffered saline solution (1% BSA / PBS). Cell suspensions were subsequently stained for flow cytometry for 30 min at 4 °C using antibodies specific for CD45 [REA747]-VioBlue and CD3 [BW264/56]-PE from Miltenyi Biotec. Cells were washed with cold PBS, and then incubated for 15 min in 1.5 mL of 1% BSA / PBS containing 1 uM calcein AM (Life Technologies) and 0.33 uM TO-PRO-3 iodide (Life Technologies). Sorting was performed with the FACS Aria Fusion Special Order System (Becton Dickinson) using 488 nm (calcein AM, 530/30 filter; CD3-PE, 585/42 filter), 640nm (TO-PRO-3, 670/14 filter), and 405 nm (CD45-VioBlue, 450/50 filter) lasers. Standard, strict forward scatter height versus area criteria were used to discriminate doublets and gate only singleton cells. Viable cells were identified by staining positive with calcein AM but negative for TO-PRO-3. We sorted individual, viable, CD45+CD3- and CD45+CD3+ immune, and CD45- non-immune single cells into 96-well plates containing cold TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen on dry ice immediately after sorting and stored at -80 °C prior to whole transcriptome amplification, library preparation and sequencing. Frozen tumor tissue (approximately 5mm x 5mm x 5mm) was placed into a well of a 6-well plate (Corning, Cat. No. CLS3516-50EA) containing 1 ml of CST buffer and processed on ice. Tumor tissue was chopped for 10 minutes using Noyes Spring Scissors (Fine Science Tools, Cat. No. 15514-12). The sample was then filtered through a 40 um cell strainer (Fisher Scientific, Cat. No. 22363547) and the well and filter were washed with an additional 1 ml of CST buffer. The total volume was then brought up to 5 ml with 3 ml of ST buffer and the sample was centrifuged in a 15 ml conical tube at 4°C for 5 minutes at 500 g. The supernatant was removed and the pellet was resuspended in 100 ul ST buffer. The sample was then filtered through a 35 um cell strainer (Falcon, Cat. No. 352235). Nuclei suspension was counted using disposable C-Chip Hemocytometers (INCYTO, Cat. No. DHCN012). Single-nuclei were loaded on 10x genomics. IdhR132H mouse glioma cells with Notch1-KO and Notch1-WT were cultured in the presence of 1 μM AG-881or vehicle (DMSO control) for > 2 weeks. The single-cell suspension for Notch1-KO and Notch1-WT mouse glioma cells treaded by AG-881 or DMSO (8,000 cells per group) were profiled by the 10X Chromium 3' Single Cell Platform and were sequenced as already mentioned. Single cells or single nucleus were processed through the 10X Chromium 3' Single Cell Platform using the Chromium Single Cell 3' Library, Gel Bead and Chip Kits (10X Genomics, Pleasanton, CA), following the manufacturer's protocol. Briefly, 8,000 cells were added to each channel of a chip to be partitioned into Gel Beads in Emulsion (GEMs) in the Chromium instrument, followed by cell lysis and barcoded reverse transcription of RNA in the droplets. Breaking of the emulsion was followed by amplification, fragmentation and addition of adapter and sample index.