3'-Seq Library Generation and Analysis

We used our recently published strategy to generate 3′-seq libraries and the computational workflow to analyze the data [29 (link)]. Briefly, 3′-seq libraries were prepared using 2 μg of total RNA as starting material. The total RNA was chemically fragmented and custom oligo-dT primers were used to capture and synthesize cDNA representing the junction of the poly(A) tail and the 3′ end of RNAs. The cDNAs were sequenced using an Illumina HiSeq-1000 sequencer with SE-50 mode. The data were mapped onto the genome assemblies and 3′ end clusters were derived and quantified within a 25-bp window as described [29 (link)].