GSM6503025: RNA-seq of E. coli wt cells in stationary phase 45°C -Replicate 3; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries. After the RNA samples passed the detection, magnetic beads with Oligo(dT) were used to enrich eukaryotic mRNA (if prokaryotes were prokaryotes, the mRNA was enriched by removing rRNA through the kit). Then mRNA was fragmented into fragments by fragmentation buffer. A strand of cDNA was synthesized by using mRNA as template and random Hexamers with six bases. Then buffer, dNTPs (dTTP in dNTP was replaced by dUTP) and DNA Polymerase I and RNase H were added to synthesize the double-stranded cDNA. AMPure XP Beads were used to purify the double-stranded cDNA, and USER enzyme was used to degrade the second strand of cDNA containing U. The purified double-stranded cDNA was repaired at the end, A tail was added, and sequencing adaptor was connected. Then AMPure XP beads were used for fragment size selection. Finally, PCR amplification was performed and AMPure XP beads were used to purify the PCR products to obtain the final library. After the construction of the library, Qubit 2.0 was used for preliminary quantification, and then Agilent 2100 was used to detect the size of the inserted fragments of the library. After the inserted fragments met the expectations, Q-PCR method was used to accurately quantify the effective concentration of the library to ensure the quality of the library.