title: GSM5366146: BH72_MinusN_2; Azoarcus olearius; RNA-Seq
library_construction_protocol: 30 mL of harvested culture was frozen in liquid N2. RNA was extracted from the frozen cells using Hot phenol method. Genomic DNA was eliminatedby using RNeasy Plus Mini Kit (Qiagen, Catalog no. 74134). Ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina, Catalog no. MRZB12424). The samples were fragmented by 4 pulses of ultrasound, 30 s each, at 4 °C. To aid in directional sequencing, 3' Illumina TruSeq sequencing adapter, with barcodes, was ligated to the 3' end of the fragmented RNA molecules. First-strand cDNA was synthesized using M-MLV reverse transcriptase and a primer binding to the adapter sequence. Synthesized cDNA was purified, and to its 3' end the 5' Illumina TruSeq sequencing adapter, also containing barcodes, was ligated. The cDNA was amplified through 13 cycles of PCR, using a high fidelity DNA polymerase, to obtain about 10-20 ng/µl of DNA. Amplified cDNA was purified using AgencourtAMPure XP kit (Beckman Coulter Genomics). The six samples were pooled in approximately equimolar amounts and size-fractionated using a preparative agarose gel to obtain the size range between 200 and 400 bp. The sample pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.