GSM5739079: PA14_DlasR_BF_R2; Pseudomonas aeruginosa; RNA-Seq
GSM5739079
The RNeasy Mini Kit (Qiagen) in combination with Qiashredder columns (Qiagen) was used according to the manufacturer's instruction Total RNA was fragmented (150-350 bp) in Fast AP-Buffer (Thermo Scientific), DNA was digested by TURBO™ DNase (Invitrogen), and RNA fragments were phosphorylated by FastAP alkaline phosphatase (Thermo Scientific). Custom-made barcodes were ligated to the RNA using the T4 ligase (New England Biolabs) and fragments were subsequently purified and concentrated using the RNA Clean & Concentrator™-25 Kit (Zymo Research) following the manufacturer's instructions. Ribosomal RNA was removed by using the RiboZero Bacteria kit (Illumina). cDNA libraries were synthesized using the SMARTScribe Reverse Transcriptase (Takara) followed by a PCR enrichment using the AccuPrime HiFi Taq polymerase (Invitrogen). Enzymatic reactions were carried out in the presence of SUPERase·In™ RNase Inhibitor (Invitrogen); RNACleanXP beads (Agencourt) were used for all RNA purification steps. Quality checks were performed before, during and after cDNA library preparation with the RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent Technologies).