GSM9303923: Tumour, control vaccine, rep4, 1 dose; Mus musculus; RNA-Seq
Tumours were excised from mice and dissociated using the Mouse Tumor Dissociation Kit (Miltenyi Biotec, 130-096-730) with the gentleMACS Dissociator (Miltenyi Biotec). CD45+ tumour-infiltrating lymphocytes (TILs) were isolated from tumours of control and vaccinated mice using Mouse CD45 TIL MicroBeads (Miltenyi Biotec, 130-110-618). The sample are biological replicates consisted of distinct tumours harvested from different tumour-bearing mice in each treatment group. Total RNA sample was extracted from isolated TILs from mice in each experimental group using the RNeasy mini-Kit (QIAGEN) per the manufacturer's protocol. RNA purity, concentration, and integrity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), Qubit 2.0 Fluorometer (Thermo Fisher Scientific), and Agilent 2100 Bioanalyzer (Agilent Technologies), respectively. Only samples meeting quality thresholds (e.g., RIN ≥ 4) were used for library preparation RNA libraries were constructed using quantified RNA samples, pooled, and sequenced on the Illumina NovaSeq 6000 platform in paired-end 150 bp (PE150) mode (Novogene), targeting a minimum depth of 6 Gb reads per sample. Raw reads were aligned to the Mus musculus reference genome (mm39, NCBI RefSeq GCF_000001635.27) using HISAT2 (version 2.0.5)