title: GSM2690479: Wild Type strain overexpressing dam, replicate 2; Photorhabdus laumondii subsp. laumondii TTO1; RNA-Seq
library_construction_protocol: RNeasy miniprep QIAGEN kit. Ribo-Zero rRNA Removal Kit Bacteria (Illumina, San Diego, CA) was used to remove ribosomal RNA from 4 µg of total RNA. For each sample, 100 ng of rRNA-depleted RNA was used to construct sequencing libraries using Illumina's TruSeq Stranded mRNA Sample Prep Kit (Low throughput). The mRNA was chemically fragmented. The first cDNA strand was generated by reverse transcription with random hexamer primers, SuperScript IV Reverse Transcriptase (Life Technologies), Actinomycine D. The second strand cDNA was synthesized by replacing dTTP with dUTP. A single ''A'' nucleotide was added to the 3' end and ligation was carried out with Illumina's indexed adapters. After 15 cycles of PCR, libraries were validated on a Fragment Analyzer (AATI, Ankeny, IA) and quantified with a KAPA qPCR kit. On a sequencing lane of a flowcell V4, nine libraries were pooled in equal proportions, denatured with NaOH and diluted to 8 pM before clustering. Clustering and 50nt single read sequencing were performed according to the manufacturer's instructions. Cluster formation, primer hybridisation and single end-read 50 cycles sequencing were performed on cBot and HiSeq2500 (Illumina, San Diego, CA) respectively.
attributes.tag: GEO Accession
attributes.value: GSM2690479