Computational protocol: Archaeal Signal Transduction: Impact of Protein Phosphatase Deletions on Cell Size, Motility, and Energy Metabolism in Sulfolobus acidocaldarius*

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Protocol publication

[…] Microscopy pictures were taken using an Axio Imager.M1 microscope (Zeiss) equipped with a Zeiss Plan Apochromat × 100/1.40 Oil DIC objective and a Cascade:1K CCD camera (Photometrics). Cell size was analyzed automatically with ImageJ and ObjectJ using a modified version of the filaments-91i.ojj project. The results were evaluated with Origin 6.1 (OriginLab Corporation, Northampton, MA, USA). [...] S. acidocaldarius MW001, Δsaci_ptp, and Δsaci_pp2a were inoculated in triplicate. Cultures were grown to reach an OD600 of 0.6 (exponential growth phase) and subsequently samples of the three independent cultures of each strain were pooled in equal amounts to generate one mixed sample per strain. Total RNA samples were isolated from 10 ml of exponentially growing shaking cultures. TRIzol reagent (Invitrogen) was used for total RNA isolation following the instructions of the manufacturer. Residual chromosomal DNA present in RNA samples was removed by RNase-free DNase I (Roche) treatment for 2 h at 37 °C. DNA-free RNA samples were confirmed by PCR amplification using saci0574 (secY) primer pairs. Before cDNA synthesis DNA-free RNA samples were fragmented to achieve a molecule size range of 50 to 500 nucleotides. Thus, 6 μg of DNA-free RNA samples were mixed with 4 μl of 5× fragmentation buffer (200 mm Tris acetate, pH 8.2; 500 mm potassium acetate; 150 mm magnesium acetate). The reaction mix was filled up with DEPC H2O up to 20 μl, incubated at 95 °C for 2.5 min and immediately transferred to ice. The reaction was then cleaned up by using sephadex columns (illustra MicroSpin™ G-25, GE Healthcare). RNA fragmentation range was checked by polyacrylamide gels. Six hundred nanograms of fragmented RNA was used for cDNA synthesis using the SuperScript Double-stranded cDNA Synthesis kit (Invitrogen) and following manufacturer's instructions. The reaction was cleaned by using phenol/chloroform/isoamyl alcohol and cDNA samples were then precipitated by using 3 m NaOAc/100% ethanol and incubated over night at −20 °C. Finally, cDNA samples were washed with 500 μl 70% ethanol. Five nanograms of cDNA were used as starting material for the generation of single-end sequencing libraries with the NEBnext DNA sample preparation kit, as described by manufacturer's protocol. The DNA was ligated to Illumina adaptors, which carried a unique four letter barcode sequence. DNA fragments of 150–500 bp were selected for sequencing. To retain strand specificity the DNA was treated with Uracil DNA-Glycosilase (UDGase) before the PCR amplification. The sequencing was performed by an Illumina Genome Analyzer IIx, multiplexed together with a total of eight samples. The reads were partitioned according to their barcode, stripped from adapter sequences and barcodes and mapped, using default settings, to the Sulfolobus acidocaldarius DSM 639 (NC_007181) reference genome, with segemehl (). [...] For each protein coding and ncRNA gene, according to the NCBI database, the associated reads were counted. An overview where the reads map to, can be found in supplemental Fig. S8A. The overall Illumina sequencing represented 26.6%, 30.2%, 26.5% of genome coverage by the mapped reads for MW001, Δsaci_ptp and Δsaci_pp2a, respectively. To identify differentially expressed genes among the sample conditions, we used DESeq (version 1.5) (), which allows estimation of the dispersion within one condition. This estimation is based on the assumption that only a few genes are truly differentially expressed; hence variance across different conditions can be seen as a too conservative estimation of the variance within one condition. From the estimated expected variance and the observed fold change in read counts, the statistical significance of altered RNA abundance can be calculated for every gene. Genes with a p value below 0.054 were considered to be significantly altered in their expression. supplemental Figs. S8B and S8C shows the correlation among the observed read counts, the fold change in read count among the sample conditions and the significance of the alteration. […]

Pipeline specifications

Software tools Segemehl, DESeq
Application RNA-seq analysis
Organisms Sulfolobus acidocaldarius
Chemicals Okadaic Acid