5 protocols
AccessionType
normalization data transformation protocol
Image analyses and basecalling were conducted using the HiSeq Control Software (HCS 1.1.37.8) and RTA component (RTA 1.7.48). Sequenced reads were mapped to the Mesorhizobium metallidurans STM2683 and STM4661 genomes using ssaha2 version 2.5.1 with parameters -solexa -s (read length)/2. samtools (version 0.1.12a (r862)) were used to discard reads corresponding to multiple matches and convert ssaha2 SAM output file to generic BAM file. Transcripts coverage (Read Count Analysis) were performed using the Genomic_Features R package (version 1.4.3) running on R (version 2.13.1). Differential Expression Analyses were performed using the DESeq R package (version 1.4.1) running on R (version 2.13.1). Genome_build: STM4661: CAAF010000001-CAAF010000089, BioProject PRJEB1502. Supplementary_files_format_and_content: Tab-delimited text files containing raw abundance measurements.
nucleic acid library construction protocol
After 5 hours incubation, 1/10th volume of ice-cold stop buffer (5% phenol in ethanol) was added to each culture and directly centrifuged at 4°C, 8,000 rpm for 4 minutes. Total RNA was extracted using the RiboPureTM kit (Ambion) following the manufacturer's recommendations. Two successive runs of ribosomal RNA subtractions were performed using the Microbe ExpressTM kit (Ambion) following the manufacturer's instructions. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 1004898 Rev. D). Briefly, the first step in the workflow involves fragmentation of mRNA into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. This was followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end-repair process, the addition of a single 'A' base, and then ligation of the adapters. These products were then purified on a gel to select a size range at 200 bp and enriched by PCR to create the final cDNA library. The efficacy of the library construction was checked in a quality control step that involved measuring the adapter-cDNA size and concentration on an Agilent DNA 1000 chip. Sequencing libraries were denatured with sodium hydroxide and diluted to 6 pM in hybridization buffer for loading onto a single lane of an Illumina HiSeq 2000 flowcell V1.5. Cluster formation, primer hybridization and single-read, 36 sequencing cycles were performed on cBot and HiSeq 2000 (Illumina, San Diego, CA), respectively. mRNAseq Part # 1004898 Rev. D Illumina Cat # RS-930-1001.
sample treatment protocol
When the pre-cultures reached the mid-exponential phase (after ca 16 hours), 15 ml were added to an equal volume of pre-warmed TY medium containing either nothing (Control treatments), Zn at 1 mM (Zn treatments, 0.5 mM final concentration) or Cd at 0.05 mM (Cd treatments, 0.025 mM final concentration) and were further incubated for 5 hours at 28°C at 145 rpm.
growth protocol
Pre-grown in Ty medium (tryptone, yeast extract and CaCl2) up to mid-exponential phase (up to OD600nm=0.5) in 250-ml Erlenmeyer flasks at 28°C and 145 rpm.
normalization data transformation protocol
Image analyses and basecalling were conducted using the HiSeq Control Software (HCS 1.1.37.8) and RTA component (RTA 1.7.48). Sequenced reads were mapped to the Mesorhizobium metallidurans STM2683 and STM4661 genomes using ssaha2 version 2.5.1 with parameters -solexa -s (read length)/2. samtools (version 0.1.12a (r862)) were used to discard reads corresponding to multiple matches and convert ssaha2 SAM output file to generic BAM file. Transcripts coverage (Read Count Analysis) were performed using the Genomic_Features R package (version 1.4.3) running on R (version 2.13.1). Differential Expression Analyses were performed using the DESeq R package (version 1.4.1) running on R (version 2.13.1). Genome_build: STM2683: CAUM01000001-CAUM01000191, BioProject PRJEB1501. Supplementary_files_format_and_content: Tab-delimited text files containing raw abundance measurements.