GSM1314512: TSS-seq (+TEX) rep1; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
rRNA-depeleted total RNA extracted from the cells cultured in M9 minimal media at 30 ℃ was used to perform dRNA-seq6,7. To remove RNA with a 5’-monophosphate end, the residual mRNA was treated by 1 U terminator exonuclease (Epicentre) and 10U SUPERase-In (Invitrogen) at 30°C for 60 min. The reaction was terminated by adding 1 μL of 100 mM EDTA (pH 8.0), followed by phenol-chloroform extraction and ethanol precipitation. The 5’-triphosphate end of primary RNA was then converted to a 5’-monophosphate end with 20 U RNA 5’ polyphosphatase (Epicentre) and 10 U SUPERase-In at 37°C for 60 min, followed by phenol-chloroform extraction and ethanol precipitation. The RNA sample was incubated with 100 pmol of 5’-RNA adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC), 20 nmol ATP, 10 U SUPERase-In, and 20 U T4 RNA Ligase at 37 °C for 3 hr. The adapter-ligated mRNA was hybridized with 100 pmol of random 3’ overhanging primer (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNN) and then converted to cDNA by 600 U SuperScript II (Invitrogen), 30 U SUPERase-In, 30 nmol dNTPs, 1 µg actinomycin D, and 100 nmol DTT at 25 °C for 10 min and 42 °C for 60 min in order. The enzymatic reaction was inactivated by incubation at 70 °C for 15 min. The residual RNAs were removed by incubation with 1 N NaOH at 65 °C for 30 min and then neutralization with 1 N HCl. A fraction of the cDNA between 100 bp and 350 bp was extracted using Pippin PrepTM (Sage science, Beverly, MA) and dissolved in 20 µL ddH2O. The cDNA was amplified using a mixture of 1 µL cDNA, 0.5 µL Phusion Polymerase (NEB), 1 µL dNTPs (10 mM each), 1 µL SYBR green (Bio-Rad), and 1 µL of P1 and P2 primer mix (25 µM each). The samples were cycled to 94 ℃ for 10 s, 57 ℃ for 20 s, and 72 ℃ for 20 s (total 13-17 cycles). The amplification was monitored and stopped at the beginning of the saturation point. A fraction of the amplified DNA between 150 bp and 400 bp was then extracted using Pippin PrepTM. A second PCR amplification was performed with 1 µL of the cDNA to a total mass up to 1 µg with 6-8 cycles. The amplified DNA libraries were then purified and subject to next-generation sequencing.
GEO Accession
GSM1314512