For microarray hybridizations, cDNA was synthesized from total RN

For microarray hybridizations, cDNA was synthesized from total RNA and directly labeled with cyanine-3-dCTP using a modification of a protocol described elsewhere

[38]. Briefly, each 50-μL reaction contained 10 μg of total RNA, 1.25 μg of random hexanucleotide primers (Promega), 100 μM each of unlabeled dATP, dGTP, and dTTP (Invitrogen), 25 μM of unlabeled dCTP (Invitrogen), 25 μM of cyanine-3-labeled dCTP (Perkin-Elmer), 25 U SUPERase•In (Ambion), and 400 U Superscript II reverse transcriptase (Invitrogen). Reactions were performed by heating at 42°C for 2 hours followed by 70°C for 10 min. RNA was then removed by adding 100 mM NaOH, heating to Momelotinib nmr 65°C for 20 min, and neutralizing with 100 mM HCl and 300

mM sodium acetate (pH 5.2). Labeled cDNA products were purified using the MinElute PCR purification kit (Qiagen) and the quantity and incorporation frequency of cyanine-3-labeled dCTP were calculated using the microarray function on a NanoDrop Spectrophotometer. Sixty ng of labeled cDNA was then loaded onto each microarray, hybridized for 17 hours at 65°C, and washed and scanned as described for labeled cRNA in the One-Color Microarray-Based Gene Expression Analysis Manual (Agilent). The fragmentation step (heating to 60°C for 30 minutes) was omitted. Hybridization signal intensities were quantified from microarray image scans using agilent feature extraction software version 9.5.3 (Agilent). Microarray data were normalized and globally scaled over the array using genespring gx software with the rma algorithm and quantile normalization [39, 40]. Mean probe signals were calculated for each of the three NVP-BGJ398 supplier biological replicates and were plotted against

their position on the ICEclc sequence Thymidylate synthase for both strands and for RNAs isolated during exponential and stationary phases. All microarray data have been deposited in the NCBI Gene Expression Omnibus http://​www.​ncbi.​nlm.​nih.​gov/​geo under accession number GSE20461. Bioinformatic tools Putative promoters, terminators and transcription factor binding sites were predicted by using the BPROM and FindTerm programs on http://​www.​Softberry.​com. The map of ICEclc was designed from SeqBuilder of the Lasergene software package (version 6.1.4, Dnastar, Inc). Acknowledgements The work of MG, MM and JvdM was supported by grants 3100A-108199 and 3100-67229 from the Swiss National Science Foundation. NP is supported by a fellowship from the Faculty of Biology and Medicin of the University of Lausanne. Electronic supplementary material Additional file 1: Supplementary tables. Location of ORFs in the ICEclc core region and bioinformatic predictions of protein function and transcription features. Primers used in this study. Probes produced for Northern hybridizations. (PDF 259 KB) References 1. Gogarten JP, Townsend JP: Horizontal gene transfer, genome innovation and evolution.

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