GSM9084107: shYT#2_2; Mus musculus; RNA-Seq
Tumor-bearing livers were minced and digested in 15 mL of 1× PBS containing 7.5 mg Pronase, 7.5 mg Collagenase P, and 1.5 mg DNase I using a C Tube (Miltenyi Biotec) and the gentleMACS Octo Dissociator (Miltenyi) for 45 minutes at 100 rpm and 37 °C. Following digestion, the cell suspension was filtered through a 100 µm cell strainer and washed with an appropriate volume of Advanced DMEM/F12 (AdDMEM/F12) medium. Red blood cell lysis was performed using the Red Blood Cell Lysis Solution (Miltenyi, #130-094-183), followed by debris removal (Miltenyi, #130-109-398) and dead cell removal (Miltenyi, #130-090-101) according to the manufacturer's protocols. Cells were then incubated with Fc receptor blocking reagent (Miltenyi) for 10 minutes on ice. Tumor cells were labeled with anti-EpCAM-APC antibody (eBioscience, #17-5791-82; 1:100) for 30 minutes on ice. After washing, cells were incubated with anti-APC microbeads (Miltenyi) for an additional 30 minutes. Labeled cells were then loaded onto LS columns (Miltenyi) and processed according to the manufacturer's instructions. Positively labeled EpCAM⁺ cells were eluted and further processed for downstream applications. For bulk RNA sequencing, total RNA was additionally purified using the RNeasy Mini Kit (Qiagen) including an on-column DNase I digestion for 15 minutes at room temperature to remove residual genomic DNA. RNA quality and concentration were assessed using the Fragment Analyzer (Agilent Technologies). For library preparation, 1 µg of high-quality RNA was used as input for mRNA enrichment using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). mRNA was then processed into sequencing libraries using the NEBNext Ultra II RNA Library Prep Kit in combination with NEBNext Multiplex Oligos for Illumina (E6440, New England Biolabs), following the manufacturer's protocol. Final libraries were quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies).
GEO
GSM9084107_r1