cDNA Synthesis From bacterial small RNA
Regulatory RNAs in prokaryotes are referred to as small RNA (sRNAs). The sRNAs are typically between 50 and 300 nt in length. We offer well established methods for the deep sequencing of bacterial small RNomes.
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Example: Preparation of cDNA from Dickeya dadantii small RNA fraction
The small RNA fraction (< 200 nt) was isolated from Dickeya dadantii total RNA (Fig. 1). From the small RNA fraction, rRNA molecules were depleted.
For enrichment of sRNA molecules carrying 5' triphosphate, one half of the sRNA preparation was further treated with Terminator exonuclease (TEX) and the other half was left untreated. Then, from both samples the 5'PPP structures were coverted to 5'P using tobacco acid pyrophosphatase (TAP).
Oligonucleotide adapters were ligated to the 5' and 3' ends of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3‘ adapter as primer. The resulting cDNA was amplified with PCR using a high fidelity DNA polymerase (Fig. B).
The cDNA libraries were paired-end sequenced (2x75 bp) on a Illumina NextSeq 500 system.
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