Total RNA was subjected to 1 round of poly(A) selection using the Poly(A)PuristTM MAG kit (Ambion), followed by fragmentation using the RNA fragmentation kit (Ambion). Deep sequencing of mRNA was based on the 3’ region extraction and deep sequencing (3’READS) method (Hoque et al. 2013) or 3'READS+ method (Zheng et al., 2016). For 3'READS, poly(A)-containing RNA fragments were isolated using the MyOne streptavidin C1 beads (Invitrogen) coated with a 5’ biotinylated chimeric U5T45 oligo (Sigma) at room temperature for 1 hr in the binding buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA), followed by washing with the washing buffer (10 mM Tris-HCl pH7.5, 1 mM NaCl, 1 mM EDTA, 10% Formamide). RNA bound to the beads was digested with RNase H (5U in 50 μl reaction volume) at 37 °C for 1 hr, which removed the poly(A) sequence and eluted RNA from the beads. Eluted RNA fragments were purified by Phenol:Chloroform extraction and Ethanol precipitation, followed by phosphorylation of the 5' end with T4 kinase (NEB). Phosphorylated RNA was then purified by the RNeasy kit (Qiagen) and was sequentially ligated to a 5’-adenylated 3’ adapter (5’-rApp/NNNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC) with the truncated T4 RNA ligase II (Bioo Scientific) and to a 5’ adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC) by T4 RNA ligase I (NEB). The resultant RNA was reverse-transcribed to cDNA with Superscript III (Invitrogen), followed by 12 cycles of PCR amplification with Phusion high fidelity polymerase (NEB). cDNA libraries were sequenced on an Illumina Hiseq 2000 sequencer.
GEO Accession
GSM2521662