title: GSM9311960: RNASeq of Pseudomonas aeruginosa UCBPP-PA14 WT 3; Pseudomonas aeruginosa UCBPP-PA14; RNA-Seq
library_name: GSM9311960
library_construction_protocol: RNeasy Plus kit (Qiagen) in combination with QIAshredder columns (Qiagen) was used according to the manufacturer's manual. Modifications:  Cell lysis was performed with 8 mg/mL lysozyme in Tris-EDTA-buffer (pH 8.0). RLT buffer with mecaptoethanol was added. Samples were frozen at -70°C for 60 min and then processed with QIAshredder columns. The remaining steps were performed according to the manufacturer's manual. After RNA elution, samples were treated with DNase (DNA-free Kit DNase Treatment & Removal, Ambion). RNA was fragmented (150–350 bp) by FastAP buffer (Thermo). End repair was performed with DNase (Thermo) and FastAP. Barcodes were ligated to each sample by T4 RNA ligase 1 (NEB). Samples were purified with the RNA Clean & Concentrator-25 Kit (Zymo). Depletion was performed according to Engelhardt., et al  (2020). cDNA libraries were generated using SMARTscribe reverse transcriptase (Clonetech). Enrichment was done by AccuPrime HiFi Taq (Thermo) and all purification steps using RNAClean XP beads (Agencourt). Libraries were sequenced in paired-end mode on an Illumina Novaseq 6000 device (2 x 50 bp)