GSM8375606: KrasG12D mutant overexpression + shLKB1, replicate3; Homo sapiens; RNA-Seq
Total RNA was extracted from the spheroids at day 7 of 3D spheroid invasion assay using a RNeasy Kit (Qiagen, Valencia, CA, USA). After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads.  First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.The quality control of library consists of three steps: (1) Qubit 2.0: tests the library concentration preliminarily. (2) Agilent 2100: tests the insert size. (3) Q-PCR: quantifies the library effective concentration precisely.