English to Russian Science Biology (-tech,-chem,micro-) иммунология

**Single-cell sorting RNA-seq analysis**

On day 8 and 11 post tumor challenge, single cell suspension of tumor was prepared by mouse tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, DE) and spleens were dissociated with gentleMACS™ Octo Dissociator (Miltenyi Biotec). Tumors and spleens from the same treatment group were pooled and viable CD8+ T cells were sorted by FACS. FACS sorted T cells were mixed with C1 Cell Suspension Reagent (Fluidigm, South San Francisco, CA) before loading onto a 5- to 10-μm C1 Integrated Fluidic Circuit (IFC; Fluidigm). LIVE/DEAD staining solution was prepared by adding 2.5 μL ethidium homodimer-1 and 0.625 μL calcein AM (Life Technologies, Carlsbad, CA) to 1.25 mL C1 Cell Wash Buffer (Fluidigm) and 20 μL was loaded onto the C1 IFC. Each capture site was carefully examined under a Zeiss microscope in bright field, GFP, and Texas Red channels for cell doublets and viability. Cell lysing, reverse transcription, and cDNA amplification were performed on the C1 Single-Cell Auto Prep IFC, as specified by the manufacturer (protocol 100-7168 E1). The SMARTer™ Ultra Low RNA Kit (Clontech, Mountain View, CA) was used for cDNA synthesis from the single cells. Illumina NGS libraries were constructed using the NEXTERA XT DNA Sample Prep kit (Illumina), according to the manufacturer’s recommendations (protocol 100-7168 E1). A total of 2,222 single cells were sequenced on Illumina NextSeq (Illumina, San Diego, CA) by multiplexed single-read run with 75 cycles. Raw sequence data (BCL files) were converted to FASTQ format via Illumina CASAVA 1.8.2. Reads were decoded based on their barcodes. Read quality was evaluated using FastQC (www.bioinformatics. babraham.ac.uk/projects/fastqc/).