Patch-seq Sequencing of mouse glioma cells
Patch-seq Sequencing of mouse glioma cells
The tissue slices were placed in a recording chamber, and individual tumor cells were targeted under a fluorescence microscope at 40x magnification. Glioma cells were distinguished based on their fluorescent labels, including GFP-labeled GL261 cells in syngeneic models and tdTomato-labeled patient-derived GSCs in xenograft models. Patch pipettes (5–8 MΩ) were filled with an intracellular solution containing 111 mM gluconic acid, 4 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 4 mM Mg-ATP, 0.3 mM Na-GTP, 5 mM Na-phosphocreatine, and 13 mM biocytin, along with 1 U/μL recombinant RNase inhibitor (Takara, 2313A, Japan). Electrophysiological recordings were performed using Quadro EPC 10 amplifier (HEKA) Patchmaster software (version 2.65). Once a high-resistance seal (≥1 GΩ) was formed, the cell membrane was ruptured to obtain the whole-cell configuration. The spontaneous EPSC of the cells were recorded when cell membrane potential were held at -70 mV. Electrophysiological data were processed using Mini Analysis Program (Version 6.0.3) and Clampfit (Version 10.4.0.36). NA NA After electrophysiological recordings, for GL261 brain slices designated for Patch-seq analysis, the cell content was extracted using negative pressure, and the contents of the pipette were immediately injected into a 0.2 mL PCR tube containing 4 μL freshly-prepared lysis buffer. The lysis buffer consisted of 1 U/μL recombinant RNase inhibitor (Takara, 2313A, Japan), 0.33% Triton X-100 (Sigma, T8787, USA), 0.5 mM dNTP (Thermo Scientific, R1121, USA), and 2.5 μM oligo-dT primer. cDNA synthesis and amplification were performed following the Smart-seq2 protocol95. RNA was extracted from single cells after electrophysiological recordings, and full-length cDNA was synthesized. The cDNA concentration was quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher, Q32851, USA), and the size distribution was determined using the Agilent Bioanalyzer 2100 (Agilent, USA). cDNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096, USA) and the Nextera XT Index Kit V2 Sets A (Illumina, FC-131-2001, USA). The libraries were pooled and sequenced on an Illumina NovaSeq 6000 instrument (Illumina, USA) with 150-base paired-end reads.