Anabaena 7120 plus PNK
The small RNA fraction (< 200 nt) was isolated from a single sample of total RNA from cultures grown under standard conditions using the RNeasy MinElute Cleanup kit (Qiagen). The preparation of cDNA libraries and sequencing was performed by the vertis AG, Freising, Germany. The small RNA fraction sample was split into two parts. One half was first treated with Antarctic Phosphatase and re-phosphorylated with T4 Polynucleotide Kinase (+ PNK), the other half was left untreated (- PNK). Then, 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 3adapter as primer. The resulting cDNAs were amplified by 12 PCR cycles using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XPkit (Beckman Coulter) and analyzed by capillary electrophoresis. The cDNA libraries were single-end sequenced with a NextSeq 500 system using 150 bp read-length.