Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display

Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. In the present data article, we have used differential display to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions. We have used pure NO donor compound detaNONOate at final concentrations of 0.1 mM and 1 mM to treat the cells for 15 min alongside control before studying their gene expression profiles. At both the treated conditions, we identified genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments. The differentially expressed genes were further analyzed in DAVID and categorized into several different pathways.


Type of data
RNA was extracted from the control and treated cells and converted to cDNA. PCR was performed using degenerate primers, products separated by denaturing poly-acrylamide gel electrophoresis. Differentially expressed transcripts were sequenced to identify the genes. Based on the data detailed pathway oriented studies could be undertaken to understand the mechanism of nitrosative stress action

Data
To identify the differentially expressed genes in the fission yeast under nitrosative stress, cells were treated with two different doses of pure NO donor i.e. 0.1 mM and 1 mM for 15 min. It was previously reported that fission yeast Schizosaccharomyces pombe cells are much more sensitive to a concentration of 3 mM of detaNONOate than to 1 mM [1,2] in terms of cell growth, lowering the mitotic index, while cell viability is maintained at 95%. The differentially expressed genes are listed in Table 1. Treatment of the wild type S. pombe cells with NO donor compound detaNONOate at a concentration of 1 mM resulted in 9 genes to be repressed while 30 genes were identified as induced ( Table 2). The differential expressions of 8 genes were confirmed by Real-Time (RT) PCR analysis. The gene expression profiles (up regulation or down regulation) obtained by differential display analysis and by RT-PCR are similar as listed in Table 3. Gene Ontology (GO) terms that were enriched in the differentially expressed gene lists were searched using the online tool DAVID. In order to identify the different pathways affected under nitrosative stress, the information provided in KEGG was referred. Genes were classified as belonging to the different pathways that were affected upon treatment with pure NO donor compound. Fig. 1 shows the 18 pathways that were significantly affected (p o0.01) when the wild type S. pombe cells were treated with 0.1 mM concentration of pure NO donor detaNONOate for 15 min at 30°C. Fig. 2 shows the 24 pathways that were significantly affected (p o0.01) when the cells were treated with 1 mM concentration of pure NO donor under similar conditions.

Chemicals
All components for the culture medium of S. pombe were purchased from Becton-Dickinson (USA). Regular laboratory reagents were purchased from the Sigma Chemical Company (USA) unless

Strains, media and treatment of the cells with NO donor compound
Wild type S. pombe (972h À ) cells were grown in a YES medium (0.5% w/v Yeast extract, 3.0% w/v Dextrose with supplements as required) at 30°C with shaking at 140 rpm. The NO donor compound detaNONOate (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate   , to ensure that the effects upon treatment are due the nitric oxide that is released into the solution and not due to any of the by-products produced by degradation of detaNONOate.

RNA isolation protocol
The protocol for RNA isolation was followed from the Nurse Lab manual available in web (http:// research.stowers.org/baumannlab/documents/Nurselab_fissionyeasthandbook.pdf). This method was used with minor modifications. The cells were harvested at 3000 rpm at 25°C. Then the cells were washed with phosphate buffered saline (PBS; pH 7.4) once. The pellet was re-suspended in 100 ml cold RNA extraction buffer (50 mM Tris-HCl; pH 8.0, 100 mM EDTA pH 8.0, 200 mM NaCl), 100 ml cold phenol:chloroform (1:1) and 5 ml 10% SDS. The tubes were vortexed vigorously after adding acid washed glass beads for 5 min at 4°C, another 200 ml RNA extraction buffer and 300 ml phenol: chloroform (1:1) were added and properly mixed by vortexing. The tubes were centrifuged at 13,000 rpm for 5 min at 4°C and the upper aqueous phase was collected. The extraction step was repeated two times with phenol:chloroform (1:1) and finally to the aqueous phase collected, ammonium acetate was added to a final concentration of 2.5 M along with 2.5 volumes of ethanol. The tubes were kept at À 20°C for overnight for RNA precipitation. The precipitated RNA was washed with 70% ethanol twice and dissolved in RNase free water. RNA was checked by measuring O.D. at 260 nm and 280 nm and their ratio after 1:500 dilutions. The concentration of RNA was calculated taking 1 O.D. correspond to 40 mg of RNA/ml.

Treatment with RNase free DNaseI
Isolated RNA was subjected to DNaseI (Thermo) treatment to exclude the possibilities of any genomic DNA contamination. To an RNase free tube the following were added: Total RNA: 1 mg; 10X reaction buffer with MgCl 2 : 1 ml; DNase I (RNase free): 1 ml (1 unit); DEPC treated water: up to 10 ml. These tubes were incubated at 37°C for 30 min. 1 ml of 50 mM EDTA was added to each of the tubes and further incubated at 65°C for 10 min. The prepared RNA (concentration 0.1 mg/ml) was used as a template for Reverse Transcriptase PCR. Thermal cycler (Applied Biosystems) was programmed as: 65°C for 5 min 437°C for 60 min 475°C for 5 min 4 4°C. After the tubes were at 37°C for 10 min, thermal cycler was paused and 1 μl MMLV Reverse Transcriptase (Thermo) was added to each tube. Then the tubes were quickly mixed well by finger tipping and the reaction was continued. After the reaction, the tubes were stored in À20°C. prepared to avoid pipetting errors. Thermal cycler was programmed as: 94°C for 30 s 440°C for 2 min 472°C for 30 s for 40 cycles followed by 72°C for 5 min 44°C. The PCR products thus obtained were radioactive and were properly stored at À 20°C.

6% denaturing polyacrylamide gel electrophoresis
6% denaturing (with 8 M urea) polyacrylamide gel was prepared in 1X Tris/Borate/EDTA buffer (TBE; pH 8.3) using Bio-Rad Laboratories' Sequencing grade gel apparatus. 3.5 μl of each sample was mixed with 2 μl of loading dye and incubated at 80°C for 2 min. These samples were loaded onto the denaturing gel. Prior to loading the samples, each well of the gel was properly flushed with the 1X TBE buffer using a sterile syringe to remove the urea from the wells. The samples were electrophoresed for about 4 h at 60 W constant power (voltage not to exceed 1700 V) until the xylene dye (slower moving) reaches the bottom. After that the power was turned off and the gel was blotted on a piece of 3 M paper. The gel was covered with a plastic wrap, and dried under vacuum on a gel dryer (Hoefer Inc., USA) at 80°C for 1 h. It is to be noted that since the DNA is acid labile, the gel should not be fixed in methanol/acetic acid otherwise it will cause difficulty in cDNA re-amplification.
The dried gel was exposed to x-ray film for 72 h; the cassette containing the dried gel and the film was placed inside a À80°C freezer to minimize diffusion and distortion of the bands and then developed. The autoradiogram was analyzed for the presence of up regulated bands in the treated lanes compared to the control and the bands of interest were marked accordingly. Using a sharp and sterile surgical blade the necessary bands of interest were excised from the dried gel after correct orientation of the gel below and the autoradiogram above and the gel pieces were stored in numbered sterile micro-centrifuge tubes at À 20°C.

Extraction and re-amplification of DNA from the excised bands of interest
The DNA contained inside the gel pieces was extracted by adding 100 ml of sterile water to each of the tubes and allowing the gel pieces to stand for 10 min, the tubes were sealed with Parafilm M (USA) and boiled in a water bath for 15 min. A short spin was given to collect all condensation and the supernatant was transferred to newly labeled tubes into which 10 ml of 3 M Sodium acetate (NaOAc), 5 ml of Glycogen (10 mg/ml) and 450 ml of absolute ethanol were added to precipitate out the DNA from solution, for quick precipitation, the tubes were placed in a À 80°C freezer for 30 min. The tubes were centrifuged at 4°C for 10 min at 10,000 rpm to collect the DNA pellet and the excess NaOAc was washed away by using 85% ethanol. Finally the DNA pellet was dissolved in 10 ml of sterile water and used for further re-amplification by PCR.
Each of the DNA bands isolated and precipitated out by the above mentioned protocol were reamplified using the same set of primers. The PCR was done in a 40 ml reaction volume according to the Core mix was prepared to avoid pipetting errors. Thermal cycler was programmed as: 94°C for 30 s 440°C for 2 min 472°C for 30 s for 40 cycles followed by 72°C for 5 min 44°C. The PCR products were run on a 1.5% agarose gel and checked for the presence of single bands.

Cloning the inserts into vector and transformation into competent cells
The PCR amplicons obtained were ligated into cloning vector pGEM-T easy (Promega). Since the multiple cloning site of this vector is located inside the coding region for the β-galactosidase gene, successfully cloned inserts will show defective translation for the β-galactosidase gene and hence those colonies will not be able to utilize X-gal as their substrate and hence produce white colonies onto IPTG/X-gal plates of Luria-Bertani Agar þAmpicillin which are selected and further grown in Luria-Bertani (LB) broth containing Ampicillin and the plasmids were isolated. The ligation protocol followed was as follows: 2X ligation buffer: 5 ml; pGEM T-easy vector (50 ng): 1 ml; DNA insert: 2.5 ml; T4 DNA ligase (3 u/ml): 1 ml. Total reaction volume was 10 ml. The tubes were mixed well and incubated at 4°C overnight for increasing the number of recombinant colonies after transformation.
The protocol followed for transformation into competent cells is hereby stated. The strain of Escherichia coli chosen was XL1 Blue from Stratagene. The lacIqZΔM15 gene on the F 0 episome allows blue-white screening for recombinant plasmids. The vials of competent cells (100 ml/tube) were taken out of À 80°C freezer and immediately set on ice. The ligated product was added to the competent cells and the tubes were set on ice for 4 h with occasional mixing by tapping gently. Heat shock was applied to the cells at 42°C for exactly 45 s and immediately set back on ice. Fresh LB broth (without antibiotic) was added to each tube in a volume of 900 ml to make the total volume 1 ml and the tubes were incubated at 37°C for 30 min with shaking at 140 rpm. Meanwhile LB agar plates containing Ampicillin at concentrations of 100 mg/ml were plated with 40 ml IPTG (0.1 M) and 40 ml X-gal (20 mg/ ml) for blue/white screening of the transformed cells. After removing the transformed cell tubes from the incubator, those cells were plated onto IPTG/X-gal containing LB agar þ Amp plates and incubated at 37°C for 12 h.

Screening of white colonies for plasmid isolation
Since the multiple cloning site in the cloning vector was in the middle of the β-galactosidase gene, successfully ligated inserts would give defective transcription of the same thus the E. coli cells harboring such plasmids would not be able to utilize the substrate X-gal present on the plates and thus would remain white while the colonies metabolizing X-gal would turn blue. Our interest thus obviously remains in the white colonies for each of our inserts. A single white colony was inoculated into 2 ml of Luria-Bertani broth containing 0.1% Ampicillin and grown at 37°C for 12 h under shaking conditions. The resulting culture was used for isolation of the plasmid containing our insert. The method used for plasmid isolation was followed as per instructions of Qiagen Mini spin Plasmid prep kit (Qiagen).
The isolated plasmids were measured spectrophotometrically at wavelengths 260 nm and 280 nm and the concentration determined by assuming that double stranded DNA with optical density 1 at 260 nm has a concentration of 50 μg/ml. Nucleic acids absorb maximally at 260 nm while proteins absorb maximally at 280 nm, the optical density ratios at 260 nm and 280 nm gives the indication of protein contamination in the plasmid preparation. The concentrations obtained for the plasmids were generally in the range of 250-300 ng/μl.

Checking for the presence of inserts by restriction digestion with EcoR1
Besides blue-white screening of transformed E. coli colonies, we checked for the presence of inserts in our isolated plasmids by digesting the plasmids with restriction endonuclease EcoRI (New England Biolabs). The protocol we followed for the restriction digestion was as follows: 10X NEB buffer: 3 ml; EcoRI (20,000 u/ml): 0.5 ml; Plasmid DNA: 1 ml; DNase free water: 25.5 ml. Total volume of the reaction was 30 ml. The tubes were incubated at 37°C in a water bath for 1 h and the products were checked on a 1.5% agarose gel.

Sequencing of the inserts inside the plasmids isolated
The inserts cloned into pGEM-T easy vector were sequenced using M13 forward and reverse primers having sequences 5 0 GTAAAACGACGGCCAGTG3 0 and 5 0 GGAAACAGCTATGACCATG3 0 respectively. The reason for cloning our inserts into such a vector prior to sequencing is that this increases the read lengths of the sequences. In our case, differential display yields fragments of genes merely 150-180 bp long which we separate on a Urea-PAGE, excise and use this DNA for the other downstream applications. We cannot afford to lose any part of our insert's sequence due to primer binding or any other reading errors, as shortening of sequences further would give low stringency results upon matching with public databases. Thus using cloning vectors provides a better option for sequencing short insert lengths as in our case where the primer binds to the vector and covers the region containing the insert and a part of the vector too.
Sequencing PCR was performed using Applied Biosystems Big Dye Terminator cycle sequencing kit v3.1 on an Applied Biosystems 9700 thermal cycler using the protocol mentioned hereby: Big dye ready mix: 1 ml; 5X reaction buffer: 2 ml; M13 Primer (Fwd or Rev) (3.2 pmol): 1 ml; DNA template (plasmid): 100 ng; DNase free water upto 10 ml. Each sample was sequenced using both forward and reverse primers and sequences matched by database searches. The program followed for the PCR was as follows: 98°C for 5 min 4(96°C for 10 s 4 50°C for 5 s 460°C for 4 min for 30 cycles) 44°C.
2.13. Sequencing PCR product purification and sequencing of purified PCR product i) Master Mix I: 10 μl MilliQ water þ2 μl of 125 mM EDTA (pH 8) (added to each tube) ii) Master Mix II: 2 μl 3 M NaOAc (pH 4.6) þ 50 μl 100% ethanol (added to each tube). 12 μl of Master Mix I was added to each of the PCR tubes containing sequencing product and the entire contents shifted to a 0.5 ml micro-centrifuge tube. 52 μl of Master Mix II was added to each of the tubes and vortexed mildly for 1 min after which the tubes were given a pulse spin. The tubes were incubated in the dark at room temperature for 15-20 min. The tubes were centrifuged at 13,000 rpm for 30 min at room temperature and the supernatant was discarded. 100 μl of 70% ethanol was added to the pellet and centrifuged at 13,000 rpm for 5 min at room temperature. The pellet was dried for 1 h in the dark at room temperature. The tubes containing the washed pellet were stored at À 80°C for sequencing.
On the day of sequencing, 20 μl of Hi-Dye Formamide from Applied Biosystems were added to each of the tubes and vortexed to dissolve the pellet completely, incubated in the dark for 15 min at room temperature. The tubes were placed on a dry bath at 95°C for 2 min and immediately placed on ice to chill. The entire contents of each tube were loaded into sequencing plate and separated by capillary electrophoresis on an Applied Biosystems 3700 sequencer.

Preparation of the differentially expressed gene lists
The DNA sequence data obtained was analyzed using Chromas Lite software by retrieving the sequences in FASTA format and matching against NCBI database using the BLAST algorithm. During the database search only the highly similar sequences were considered. Since only the differentially regulated transcripts which were identified from the autoradiogram were excised and further processed, identification of the genes from database searches allowed us to build the list of the differentially expressed genes under nitrosative stress.

Validation of differential gene expression by Real Time PCR analysis
The cell growth, stress conditions and RNA extraction methods have been previously described.
The first strand cDNA was synthesized using 400 ng of total RNA in a 20 μl reaction volume, using the Verso cDNA synthesis kit (Thermo). The primers for the genes (Table 4) whose expression was checked were added to a final concentration of 600 nM in a total reaction volume of 50 μl containing 25 μl of Power SYBR Green PCR master mix (Applied Biosystems). The reference housekeeping gene considered was act1 coding for Actin. The instrument used was the 7300 Real-Time PCR System (Applied Biosystems). All the results were generated from two independent biological repeats and for each biological experiment three technical repeats were performed. Relative quantification of gene expression was performed by the 2 À ΔΔCt method as described in a previous report [3]. The PCR conditions consisted of denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s, and continued extension at 72°C for 5 min.

Identification of the affected pathways from the differentially expressed genes
Online tool DAVID (http://david.abcc.ncifcrf.gov/) [4,5] was used to find out the affected pathways among the differentially expressed gene lists. The gene lists were uploaded with selecting the background as all the genes of S. pombe. Functional Annotation Chart was visualized using the p-value threshold o 0.01 and count 2. The information regarding the affected pathways was obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG) within the analysis in DAVID, using the mentioned thresholds. Table 4 Primer sequences of the genes validated by Real-Time PCR.