GSM4508588: 100 DBN R2; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
Trizol total RNA extraction from E. coli was performed according to the manufacturer's protocol. (1) Poly adenylation. 100 ng of total RNA in 2 µL was combined with 3 µL poly(A) mix, comprised of 1 µL ThermoFisher 5x first strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2], 1 µL blocking primer mix (50 µM), 0.8 µL nuclease-free water, 0.1 µL 10 mM ATP, and 0.1 µL E. coli poly(A) polymerase. The mixture was incubated at 37°C for 10 min. In the control group, no blocking primers were added and 1.8 µL of nuclease-free water was added instead. The blocking primer mix was prepared by mixing equal volumes of 50 µM blocking primers specific to 5S, 16S, and 23S rRNA. (2) Reverse transcription. The polyadenylation product was mixed with 0.5 µL 10 mM dNTPs, 1 µL RT primers (25 ng/µL), and 1.3 µL blocking primer mix (50 µM), and heated to 65°C for 5 min, 58°C for 1 min, and then quenched on ice. In the control samples, the blocking primers were again replaced with nuclease-free water. Next, 3.2 µL RT mix, consisting of 1.2 µL 5x first strand buffer, 1 µL 0.1 M DTT, 0.5 µL RNaseOUT, and 0.5 µL Superscript II reverse transcriptase was added to the solution, followed by 1 h incubation at 42°C. The temperature was then raised to 70°C for 10 min to heat inactivate Superscript II. (3) Second strand synthesis. 49 µL of the second strand mix, containing 33.5 µL water, 12 µL 5x second strand buffer [100 mM Tris-HCl (pH 6.9), 23 mM MgCl2, 450 mM KCl , 0.75 mM β-NAD, 50 mM (NH4)2 SO4], 1.2 µL 10 mM dNTPs, 0.4 µL E. coli ligase, 1.5 µL DNA polymerase I, and 0.4 µL RNase H, was added to the product from the previous step. The mixture was incubated at 16°C for 2 h. cDNA was purified with 1x AMPure XP DNA beads and eluted in 24µL nuclease-free water that was subsequently concentrated to 6.4 µL. (4) In vitro transcription. The concentrated solution was mixed with 9.6 µL of Ambion in vitro transcription mix (1.6 µL of each ribonucleotide, 1.6 µL 10x T7 reaction buffer, 1.6 µL T7 enzyme mix) and incubated at 37°C for 13 h. Next, the aRNA was treated with 6 µL EXO-SAP at 37°C for 15 min followed by fragmentation with 5.5 µL fragmentation buffer (200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc) at 94°C for 3 min. The reaction was then quenched with 2.75 µL stop buffer (0.5 M EDTA) on ice. The fragmented aRNA was size selected with 0.8x AMPure RNA beads and eluted in 15 µL nuclease-free water. Thereafter, Illumina libraries were prepared as described previously in Hashimshony et al. 2016 Genome Biology.
GEO Accession
GSM4508588