Total RNA was isolated using TRIzol (Invitrogen) and digested with DNase I. cDNA libraries were constructed by Vertis Biotechnologie AG (Freising) using an in-house protocol. Total RNA (DNase I digested) was fragmented by ultrasound (4 pulses of 30s at 4°C). 3′ ends were subjected to poly(A)-tailing using poly(A) polymerase, pyrophosphate groups were removed from the 5′ terminus using tobacco acid pyrophosphatase (TAP), and an RNA adapter was ligated to the 5′ end of the RNA. First-strand synthesis was performed using an oligo(dT) adapter and M-MLV-RNaseH-reverse transcriptase, followed by PCR amplification of cDNA using high-fidelity DNA polymerase to ∼20–30 ng/μL. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis.
GEO Accession
GSM2091448