title: TCRb-seq of mouse CD8+ T cells isolated from Cd4-Cre and Ch25hfl/flCd4-Cre mice following VSV-OVA infection
design: TCRb-seq of mouse CD8+ T cells isolated from Cd4-Cre and Ch25hfl/flCd4-Cre mice following VSV-OVA infection
library_construction_protocol: KLRG1+CD127- effector cells were sorted from OVA257-264-H-2Kb tetramer-binding CD8+ T cells isolated from Ch25hfl/fl and Ch25hfl/flCd4-Cre mice after infection with VSV-OVA. Cell sorting was performed on a BD FACSAria IIu flow cytometer. Following VSV-OVA infection, mice were euthanized via CO2 asphyxiation. Spleens were aseptically dissected. A single-cell suspension was prepared from the splenic tissue for subsequent staining and cell sorting. Mice were housed under a 12-hour light-dark cycle, at temperatures of 18–22°C, and at a humidity of 30–70%. Mice were infected intravenously with 1 × 10^6 plaque-forming units (PFU) of recombinant VSV-OVA. Image analysis and base calling were conducted by the MiSeq Control Software (MCS) or NovaSeq Control Software (NCS) on the MiSeq/NovaSeq instrument, converting the raw signal data into demultiplexed FASTQ files. Total RNA was extracted from the sorted KLRG1+CD127- effector cell samples using TRIzol reagent. Libraries were constructed from total RNA using a 5' RACE approach for reverse transcription, followed by TCR-specific PCR amplification. The PCR products were purified, end-repaired, and ligated to sequencing adapters. The final libraries were amplified with P5 and P7 primers, purified, and quality was assessed using a Qsep100 and Qubit 3.0 Fluorometer.