Single-cell RNA-seq analysis of syngeneic tumors treated with intratumoral NHS-rmIL12 immunotherapy
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ABSTRACT: Established MOC22 tumors in WT B6 mice were treated with three intratumoral injections of 0.4 ug NHS-rmIL12; control and treated tumors were analyzed for immune correlative changes with single-cell RNA-seq.
Project description:To compare total RNA levels in miR-124 and mock-transfected cells (Figure S3), 5-10 ug of total RNA from miR-124-transfected cells or mock-transfected cells or universal reference RNA (Stratagene Cat# 740000) was reverse transcribed with Superscript III (Invitrogen Cat# 18080085) in the presence of aminoallul-dUTP 5-(3-aminoallyl)-dUTP (Ambion Cat# AM8439) and natural dNTPs (GE Healthsciences Cat# US77212) with 10 ug of N9 primer (Invitrogen). Subsequently, amino-allyl-containing cDNAs from miR-124 and mock-transfected cells were covalently linked to Cy5 NHS-monoesters, and universal reference cDNA was covalently linked to Cy3 NHS-monoesters (GE HealthSciences Cat# RPN5661). Cy5- and Cy3-labeled cDNAs were mixed and diluted into 50 ul of solution containing 3x SSC, 25 mM Hepes-NaOH (pH 7.0), 20 ug human Cot-1 DNA (Invitrogen Cat# 15279011), 20 ug of poly(A) RNA (Sigma Cat# P9403), 25 ug of yeast tRNA (Invitrogen Cat# 15401029), and 0.3% SDS. The sample was incubated at 95 C for 2 min, spun at 14,000 rpm for 10 mins in a microcentrifuge, then hybridized at 65 C
Project description:Surface shaving Mtb using two concentrations of trypsin: 0.4 ug/mL and 0.05 ug/mL. Both bacteria and supernatant without the bacteria were subject to trypsinization, to allow for a ratio between samples to identify candidate surface proteins.
Project description:To detect gene expression changes in skeletal muscle caused by EVs from mMCF-10A/miR-122 cells, we analyzed RNA isolated from the GA muscle of EV-treated female NOD scid gamma (NSG) mice. Mice had received tail-vein injections of EVs twice a week for 5 weeks (~10 ug EV per injection). Gene expression in muscle from mice treated with MCF-10A/miR-122-derived EVs.
Project description:To compare gene expression changes in skeletal muscle caused by EVs from normal (MCF-10A) and cancer (MDA-MB-231) cells, we analyzed RNA isolated from the GA muscle of EV-treated female NOD scid gamma (NSG) mice. Mice had received tail-vein injections of EVs twice a week for 5 weeks (~10 ug EV per injection). Gene expression in muscle from mice treated with MDA-MB-231-derived EVs was compared to mice treated with MCF-10A-derived EVs.
Project description:Expression of genes in sorted CD4, CD8 and non-T CD45 cells isolated from TME of B16 tumors in wildtype B6, APR-246 treated wildtype B6 and Super p53 mice.