48 channel fragment analyzer

One million cells with viability of over 90% by Trypan blue measurement were collected, centrifuged, washed in PBS, and lysed in RLT buffer (79216, Qiagen) before storage at -80°. RNA was later extracted using the Qiagen RNeasy Mini Kit (74104, Qiagen, United States) according to manufacturer’s specifications. An on-column DNA digestion (DNASE10-1SET, Sigma) was performed; and the final elution volume was 30 μl. Total RNA was quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Q33140, Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit (DNF-471-0500, Agilent) on a 48-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 25 ng/μl, while RNA integrity number (RIN) ranged from 8.2 to 10, with a median of 9.9.

HCT116^WT and HCT116^CD276KO cells were cultured and maintained in different culture flasks for further propagation. Five biological replicates were used per group for a total of 10 samples. 1 × 10⁶ cells per sample were collected, centrifuged, washed two times with PBS. After removal of supernatant completely, cell pellets were immediately frozen at − 80 °C until further use. Total RNA extraction for Next-Generation Sequencing from cell pellets lysed in RLT plus buffer were performed by using the AllPrep DNA/RNA Mini Kit (80224, Qiagen) according to the company’s protocol. An on-column DNA digestion was performed, and the final elution volume was 30 µL.
Total RNA was quantitatively and qualitatively assessed using the fluorescence-based RNA Broad Range Qubit Kit (Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 48-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 30 ng/µL while RIN were all > 8.5.