RNA-seq profiling in metachronous colorectal liver metastasis
RNA-seq profiling in metachronous colorectal liver metastasis
Metachronous colorectal liver metastasis tissue samples and their adjacent non-malignant liver tissues surgically resected from patients.  Total RNA was extracted from tumor tissue using the TRIZOL reagent (Thermofisher) following the manufacturer's protocol. The tissue was first pulverized with mortar and pestle under liquid nitrogen.  500 ng of total RNA was used for construction of RNA libraries. The cDNA libraries corresponding to mRNA were constructed using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen), following the manufacturer's protocol. Samples with RIN>3.5 (n=72) were processed by the standard protocol, while samples with RIN<3.5 (n=14) were processed using the Low Quality RNA protocol. Quality of prepared libraries was checked by Tapestation 2200 using High Sensitivity D1000 kit (Agilent Technologies Inc., CA, USA) and quantity measured by the Qubit instrument using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, MA, USA). Prepared libraries were pooled equimolarly.