title: scRNA-seq of MSK-115 CD45+ Sorted IDH-MUT Glioma Sample
design: Tumor tissue from glioma operations was collected immediately after resection and transported in MACS storage solution (Miltenyi Biotec, Germany) within 15 min to the laboratory. Resected tissue was morselized into small pieces under sterile conditions using sterile scalpels, triturated using pipettes, then transferred to a 50 mL falcon tube (Corning, USA) for incubation with papain (37C, 25 min) (Worthington, USA) according to manufacturer instructions. After incubation HBSS without magnesium or calcium was then added to make up to 50 ml and the sample was centrifuged (300g, 5 min, 4C) and supernatant aspirated before being passed through a 70um cell strainer (BD Biosciences, USA). Erythrocytes were removed by resuspending and incubating the obtained pellet in 5 ml of ACK-lysis buffer (eBioscience, USA) for 10 min, followed by centrifugation (300g, 5 min,4C). Residual myelin and extracellular debris were eliminated using the Debris Removal Kit (Miltenyi Biotec, Germany). Cell counts were determined using an automated cell counter (Nexcelom Bioscience, USA) after resuspending the pellet in PBS. A portion of the single-cell suspensions were then enriched for CD45+ cells by CD45-MACS enrichment (Miltenyi Biotec, Germany). The final cell suspensions were centrifuged again (350g, 10 min, RT) and isolated unsorted, CD45+, and CD45- cells were stored in Bambanker (Wako, Japan). Cell suspensions were immediately placed in a storage container (Mr. FrostyTM, ThermoFisher Scientific, USA) and stored in LN2. Single cells from each sample were split into two populations to undergo single-cell RNA- and ATAC-sequencing, respectively. For scRNA-seq, cells were loaded for GEM generation using the 10x Genomics Chromium platform. 3 RNA gene expression libraries were prepared using the Chromium Single Cell 3 Reagent Kit v3 (10x Genomics, USA) according to the manufacturers guidelines. Final libraries were quantified using a Qubit fluorimeter (Life Technologies, USA) and the nucleosomal pattern was verified using a BioAnalyzer (Agilent Technologies, USA). scRNA  libraries were sequenced on a NovaSeq6000 (Illumina, USA). Raw sequencing reads were aligned to the GRCh38 reference genome using Cell Ranger (v7.1)