alias: GSM3530860
title: GSM3530860: PA14 WT+ HOCl 1; Pseudomonas aeruginosa; RNA-Seq
library_construction_protocol: After 20 minute incubation with HOCl or HOSCN, cells were harvested, washed and RNA preserved by addition of RNAlater (Ambion). Bacteria were lysed with lysozyme and proteinase K buffer and total RNA extracted using the RNeasy Protect Bacteria Mini Kit (Qiagen), according to the manufacturer's instructions. RNA samples were DNase treated using a TURBO DNA-free kit (Ambion), according to the manufacturer's instructions, prior to sending RNA on dry ice to vertis Biotechnologie AG for preparation of cDNA libraries and Illumina sequencing. rRNA was removed using the bacterial Ribo-Zero rRNA Removal Kit (Illumina), and then RNA fragmented by ultrasound. An oligonucleotide adapter was ligated to the 3' end of the RNA and first-strand cDNA synthesis carried out with M-MLV reverse transcriptase. The cDNA was purified and a 5' Illumina TruSeq sequencing adapter (with a TruSeq barcode sequence) was ligated to the 3' end of the antisense cDNA. PCR amplification of the cDNA to about 10-20 ng/µl was carried out with a high-fidelity DNA polymerase for 11 or 12 cycles. The cDNA was purified and then pooled and size fractionated in the range of 200-500 bp. The cDNA pool was single-read sequenced on an Illumina NextSeq 500 system using a 75 bp read length.
identifiers.namespace: GEO
identifiers.id: GSM3530860