title: HiSeq X Ten paired end sequencing; Genome-wide profiling reveals cancer-related genes have switched alternative polyadenylation sites in colorectal cancer
design: Genome-wide profiling reveals cancer-related genes have switched alternative polyadenylation sites in colorectal cancer
library_construction_protocol: Fresh tissues from 3 colorectal cancer patients were collected at the Anhui medical university first affiliated hospital, Anhui, China. All samples were examined by one experienced pathologist. After collected, samples were put in liquid nitrogen quickly, and preserved in a low temperature environment. Liquid nitrogen quick-frozen tissues were used to extract total RNA. Total RNA was extracted using TRIzol reagent (Invitrogen) and resuspend in RNase free water. Total RNA was extract with TRIzol Reagent . And about 50 μg total RNA was incubated with streptavidin dynabeads coated with biotinylated GsuI-oligo (dT) primer. The first strand cDNA was synthesized with SuperScript III (Invitrogen) and dNTP mix containing 5-methylated-dCTP instead of dCTP. Then the resulting DNA/RNA hybrids were treated with RNase H and DNA polymerase I before dsDNA was generated and dNTP mix containing dUTP instead of dTTP was used in this step. Next, the resulting dsDNA was fragmentated to ~200–500 bp with fragmentase (NEB). 3' terminal fragments were released from beads through Gsu I digestion, which were subjected to deep sequencing by Illumina HiSeq X Ten.