GSM8633852: tuNK, rep4; Mus musculus; RNA-Seq
Tissues (tumor and spleen) were dissected and dissected tissue were mechanically minced and digested through stirring in RPMI 1640 medium that contained collagenases Type IV (1 mg/mL, Thermo Fisher Scientific), dispase II (100 μg/mL, Sigma-Aldrich), hyaluronidase (100 μg/mL, Sigma-Aldrich), and DNase I (100 μg/mL, Sigma-Aldrich) at 37 °C for 60 min. Red blood cells were removed with ACK lysing buffer after digestion. TILs from tumor were enriched by density gradient centrifugation against Percoll (GE Healthcare) and resuspended in FACS buffer (1 × PBS containing 0.2 % BSA (wt / v), Sigma-Aldrich). Splenocytes and TILs were sorted by MACS (Miltenyi) and FACS (Sony). Total RNA extraction was carried out using the RNeasy Mini Kit (Qiagen), followed by quality assessment using TapeStation 4200 (Agilent Technologies) to ensure RNA integrity (scores > 7). High-quality RNA samples were then stored at −80°C until library preparation. Libraries for mRNA-seq were prepared utilizing the NEBNext Ultra II Directional RNA Library Prep with PolyA method (NEB), starting from 20 ng RNA, in accordance with the NEBNext Ultra II Directional RNA Library Prep Kit, Version 4.0_4/21 (internal exp code CSX063). Each library, distinguished by unique dual indexes, was subsequently loaded onto a NovaSeq 6000 flow cell (Illumina) and sequenced following the manufacturer's recommendations, yielding at least 40 million pairs of 60 nucleotides-long reads per sample.