title: GSM8491074: post_SC_3; Mus musculus; RNA-Seq
library_construction_protocol: Tumor tissues, subcutaneously and hepatically grown for 15 days, were excised, minced, and subjected to a digestion solution containing 100 mg collagenase, 5 mg DNase, and 5 mg hyaluronidase in 50 ml DMEM, enriched with 1 ml FBS. This mixture was incubated at 37°C for 45 minutes. Post-digestion, the suspension was filtered and centrifuged at 4°C to isolate the cells. The resulting subcutaneous (SC) and intrahepatic (Liver) tumor cells were resuspended in complete DMEM and stored in liquid nitrogen. Total RNA was isolated from frozen tumor cells using Trizol reagent (Thermo Fisher, USA) according to the manufacturer's instructions. The integrity and concentration of the extracted RNA were assessed with a Bioanalyzer 2100 (Agilent, USA), and only samples with an RNA Integrity Number (RIN) greater than 7.0 were used for further experiments. Poly(A) mRNA was purified from 1 μg of total RNA using Dyna beads Oligo (dT) (Thermo Fisher, USA) through two rounds of purification. The purified mRNA was then fragmented into smaller pieces by exposure to elevated temperatures and divalent cations. Following the protocol outlined in the mRNA-seq sample preparation kit (Illumina, USA), the fragmented RNA was reverse transcribed to construct the cDNA library. The final cDNA library had an average insert size of approximately 300 bp (±50 bp).