Poly(A) Tag Library Construction from 10 ng Total RNA

Abstract

Alternative polyadenylation has been demonstrated as a tier of gene expression regulation in eukaryotes. However, its role has not been elucidated at the cellular level. Equipped with techniques to isolate single cells by fluorescence-activated cell sorting (FACS) and laser captured micro-dissection, analysis of alternative polyadenylation in specific cell types becomes possible. We present a method to generate poly(A) tags for high-throughput sequencing (PAT-seq) libraries from very low amount of total RNA. This protocol targets the junction of the 3′-UTR and poly(A) tail of transcripts. Ten nanograms of total RNA isolated from the FACS-sorted cells was reverse-transcribed to double stranded cDNA with a anchored oligo dT₍₁₈₎ primer containing maximal T7 promoter sequence. Then, an RNA amplification step using in vitro transcription of T7 RNA polymerase was carried out. Achieved cRNA was fragmented by partial digestion. First strand synthesis was carried out by using a partial adaptor sequence with random 9-nt primer to introduce the adaptor at the 5′ end. An anchored oligo dT primer containing adaptor sequence on 3′ end was introduced through second strand cDNA synthesis. This new method has been applied to investigate polyadenylation using nanogram amount of total RNA from Arabidopsis cells.