GSM8131778: DMSO_P1_A09_S105, Smart-seq2 (multi-omics); Mus musculus; RNA-Seq
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 or stained by Ruby (ThermoFisher, V10309) and sorted into 96 well plates. IdhR132H mouse glioma cells were cultured in the presence of 1 μM AG-881or vehicle (DMSO control) for > 2 weeks.  The treated cells were washed with cold PBS and then stained with 1 uM calcein AM (Life Technologies) and 0.33 uM TO-PRO-3 iodide (Life Technologies) for 30 min at 4°C. Sorting was performed with the FACS Aria Fusion Special Order System (Becton Dickinson) using 488 nm (calcein AM, 530/30 filter) and  640nm (TO-PRO-3, 670/14 filter). Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets. Calcein AM positive and TO-PRO-3 negative live cells were sorted into 96 wells containing TCL buffer (QIAGEN) for the single-cell multi-omics experiment. RNA from single cells or single nucleus was first purified with Agencourt RNAClean XP beads (Beckman Coulter). B65Libraries from isolated single cells were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications.oligo-dT were used to prime reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 768 cells with unique barcodes were combined and sequenced using a NextSeq sequencer (Illumina). Multi-omics library (DNA methylome and transcriptome) was generated based on the Smart-RRBS protocol (Chaligne et al, 2021).