GSM8641617: EAC030_Tumor_Tibia_GEX; Mus musculus; RNA-Seq
Age-matched 6-8 week-old EPZFTA-RELA and NestinCreERT2 mice were intravenously injected with CD45-PE 3 minutes prior to schedule 1. For skulls the dura was peeled and removed with fine forceps. Both the tibia and skull were then cut into small pieces using sterile scissors and mechanically dissociated in FACS buffer with a pestle, followed by a filtration step through a 70-μm cell strainer. Samples were centrifuged for 5 minutes at 420 x g. For peripheral bone marrow, both tibia were flushed with 0.05% BSA PBS with 0.05% EDTA, filtered through 7- µm meshes and washed with 2% fetal bovine serum in RPMI and resuspended on 0.05% BSA PBS solution. The whole intact deep cervical lymph nodes were mashed through a 70 μm cell strainer, using a sterile syringe plunger, and washed with 5 mL of FACS buffer. Deep cervical lymph node samples were then filtered through 70 µm meshes. Samples were centrifuged for 5 minutes at 420 x g.. Brains were macrodissected based on TdTomato fluorescent signal to harvest the tumor and region-matched brain in non-tumor bearing animals. Tumor/brain samples were mechanically dissociated using sterile surgical scalpels into ∼1 mm3 cubes, and dissociated using the mouse tumor dissociation kit (Miltenyi Biotec) using the gentleMACS Octo Dissociator (Miltenyi Biotec). After dissociation, the samples were then filtered through 100 µm meshes and washed with 2% fetal bovine serum in RPMI, spun down 420 g for 5 minutes. Samples were resuspended in 40% percoll and centrifuged at 600 x g for 10 minutes. Samples were stained with DAPI (0.2 µg/ml). Samples were centrifuged, resuspended in FACS buffer with anti-CD16/32 (FC block; Biolegend) diluted 1:50 in FACS buffer and fluorescently conjugated antibodies (anti-CD45 APC and anti-Ter 119 FITC)  at 4°C, followed by for 20 minutes. Cells were sorted using the Influx Cell Sorter (BD Biosciences) or FACsAria II (BD Biosciences) into 1% BSA coated 1.5 mL Eppendorf tubes with 500 μL of DMEM. Single-cell gene expression (GEX) and TCR/BCR libraries were constructed using the 10x Genomics Chromium platform. Cells were partitioned into Gel Bead-In-Emulsions (GEMs) with barcoded oligonucleotides for cell and molecular indexing. For GEX libraries, mRNA was reverse-transcribed, cDNA was amplified, and sequencing libraries were prepared. For TCR/BCR libraries, cDNA was enriched for V(D)J regions using target-specific primers before library preparation. Libraries were indexed, quantified, and sequenced on an Illumina platform. Paired-end sequencing enabled capture of transcriptomes and full-length V(D)J sequences, linking receptor information to gene expression profiles for each cell. Data were processed with 10x Genomics Cell Ranger pipelines.