GSM1462579: ∆ssrA RNA-seq; Salmonella enterica subsp. enterica serovar Typhimurium str. ST4/74; RNA-Seq
Total RNA was isolated using TRIzol (Invitrogen) and Dnase I digested 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). The to construct dRNA-seq libraries, these RNA samples were subsequently digested with Terminator Exonuclease (TEX). 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, following 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.