Global mapping of polyadenylation site use in proliferating and quiescent (contact-inhibited and serum-starved) cells

Purpose: To determine genes that undergo alternative polyadenylation in proliferating versus quiescent fibroblasts. Methods: Three different biological replicates (fibroblast strains, 10-1, 12-1, and 12-2) were used for generating proliferating and quiescent (7-day contact inhibited and 7-day serum-starved) cells. RNA extracted from these cells were used for library preparation. cDNA fragments were enriched for the junction between the poly(A) site and the end of the 3' UTR using a modified high throughput sequencing protocol (GnomeGen). cDNA libraries were created according to Gnome-Gen RNA-seq library preparation kit for RNA profiling except Amgen Ampure XP beads were used instead of the Gnome-gen size selector product to remove ligation reaction products before proceeding to the reverse transcription step. The libraries were sequenced on an Illumina HiSeq 2000 instrument. The sequencing reaction wasa run for 147 cycles. Reads from pol(A) enriched cDNA libraries were aligned to the genome using the STAR alignment algorithm after the computational removal of untemplated adenosines. Results: Polyadenylation site selection was significantly altered in approximately 10% of genes in quiescent compared with proliferating fibroblasts. Contact inhibited and serum starved fibroblasts had similar polyadenylation site selection profiles. Conclusions: Quiescence is associated with changes in polyadenylation site selection. Overall design: Three samples of proliferating fibroblasts, three samples of contact inhibited fibroblasts and three samples of 7-day serum starved fibroblasts were analyzed with polyadenylation site-enriched RNA-Seq.