GSM5122995: OSM4.2_B02_S686; Mus musculus; RNA-Seq
For mice samples, collected brains were mechanically and enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec) supplemented with 0.1% typeI collagenase (Thermo Fisher Scientific) / PBS. The dissociated cells were first stained with calcein Blue AM (Thermo Fisher Scientific) and Zombie NIR (BioLegend) for 25 min at 4 ℃, and with anti-mouse CD16/32 (BD Biosciences) for 5min. After washing cells with ice-cold 1% BSA / PBS, the cells were then stained with a combination of following antibodies; PerCP anti-mouse CD45 (clone 30-F11, BD Biosciences), phycoerythrin (PE) anti-mPdgfra (clone APA5, BioLegend), Allophycocyanin (APC) anti-mPodoplanin (clone 8.1.1, BioLegend), and APC anti-CD11b (clone M1/70, BioLegend), for 30 min at 4 ℃. Sorting was performed with Becton Dickinson Influx cytometer (Becton Dickinson). Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein blue AM positive and Zombine NIR negative. We sorted viable CD45 negative/ GFP positive or CD45 positive/ CD11b positive single-cells into 96-well plates containing 5uL of TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen immediately after sorting and stored at -80 ℃ prior to whole transcriptome amplification, library preparation and sequencing. Libraries from isolated single cells from mouse model were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications. RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter) before oligo-dT primed 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 with paired-end, 38-base reads, using a NextSeq 500 sequencer (Illumina). Comma-seperated file containing TPM values (transcript-per-million reads) for 6608 genes across 679 cells. For the cell lines, scRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit v2 and the 10x Chromium Controller (10x Genomics) according to the 10X Single Cell 3' v2 protocol. The cell lines were processed as a pool that consisted of 6 total cell lines, 4 of which were used in this study (MGG23, MGG75, MGH143, MGG18). Two of the used cell lines, MGG23 and MGH143 were profiled again in a different pool. Briefly, we generated a single cell suspension of the pool in 0.04% PBS-BSA (95% viability) and loaded approximately 10,500 single cells to the Chromium Controller with a targeted recovery of 6,000 cells. Single cells, reagents and single gel beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized droplets and subjected to reverse transcription. Droplets were broken and the barcoded cDNAs were purified with DynaBeads and amplified by 12 cycles of PCR (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 12; 72°C for 1 min). The amplified cDNA was fragmented, end-repaired, ligated with index adaptors, and size-selected with clean-ups between each step using the SPRIselect Reagent Kit (Beckman Coulter). Quality control of the resulting barcoded libraries was performed with the Agilent TapeStation and by PCR with primers specific to the P5 and P7 sequence (NEBNext Library Quant Kit for Illumina, New England Biolabs). single cell RNA-seq