GSM4815482: Z1615_M9_Rep3; Escherichia coli O157; RNA-Seq
Total RNA was extracted using a mirVana™️ microRNA extraction kit Total RNA samples were purified and concentrated using the Agencourt RNAClean XP kit (Beckman-Coulter). Ribosomal RNA molecules were depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina). The ribodepleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C) and oligonucleotide adapters were then ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high-fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. Purified cDNA was pooled and sequenced on an Illumina NextSeq 500 system using 75 bp read length.
GEO
GSM4815482