|
| Status |
Public on Sep 07, 2007 |
| Title |
Infected cells series 2, t=3 h after infection (inf_3h_rep2) |
| Sample type |
RNA |
| |
|
| Source name |
Infected cells series 2, t=3 h after infection
|
| Organisms |
Prochlorococcus marinus subsp. pastoris str. CCMP1986; Tiamatvirus PSSP7 |
| Characteristics |
Infected cells
|
| Treatment protocol |
Prior to phage or spent medium addition, cells were concentrated to 108 cells·ml-1 by centrifugation (10,000 Xg for 10 min at 20 °C). The cultures were split into two subcultures. Just prior to t=0 h, phage or spent medium were added to the appropriate subcultures. Samples at different time points after treatment addition were collected by centrifugation (12,400 Xg for 15 min at 20 °C), resuspended in storage buffer (200 mM sucrose, 10 mM sodium acetate pH5.2, 5 mM EDTA), snap frozen in liquid nitrogen and stored at -80 °C. Prior to RNA extraction the samples were thawed on ice and the storage buffer was removed after spinning the cells for 2 min at 20,000 Xg at 20 °C.
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| Growth protocol |
Prochlorococcus MED4 was grown in the Pro99 seawater based medium amended with 10 mM HEPES (pH7.5) and 12 mM sodium bicarbonate at 21 °C under continuous white light at 25 µmol photon·m-1·s-1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Ambion’s mirVana RNA isolation kit according to manufacturer’s instructions, after resuspending the pellet in 600 µl lysis/binding buffer from the kit. DNA was removed by DNase I digestion using the Turbo DNA-free kit (Ambion) according to manufacturer’s instructions. Eight µg of the nucleic acid extract was digested with 6 U of Turbo DNase during a 60 min incubation at 37 °C followed by DNase I inactivation with inactivation slurry. The RNA was purified and concentrated by sodium acetate/ethanol precipitation. DNA removal was verified by gel electrophoresis.
|
| Label |
biotin
|
| Label protocol |
Synthesis of complementary DNA (cDNA), labeling, hybridization, staining and scanning was carried out according to Affymetrix protocols for E.coli with minor changes. Total RNA (2 µg) was denatured at 70 °C and annealed to random hexamer primers (25 ng/µl) at 25 °C for 10 min. The RNA was reverse transcribed to produce cDNA with Superscript II (25 U/µl – Invitrogen Life Technologies) and 0.5 mM dNTPs in the presence of 1 U/µl RNase Out RNase Inhibitor (Invitrogen). The mix was incubated at 25 °C for 10 min followed by 60 min incubations at 37 °C and 42 °C respectively. Superscript II was inactivated with a 10 min incubation at 70 °C. Sodium hydroxide (0.25 N) was used to remove RNA during a 30 min incubation at 65 °C, followed by neutralization with HCl. The cDNA was purified with MinElute PCR purification columns (Qiagen). Fragments of cDNA, 50-200 nt long, were produced from a 10 min incubation at 37 °C with DNase I (0.6 U per µg cDNA), followed by heat inactivation of the DNase I enzyme (10 min at 98 °C). The cDNA fragments were end-labeled with biotin using the BioArray Terminal Labeling Kit (Enzo) during a 60 min incubation at 37 °C. The reaction was stopped by freezing at –20 °C. The quality of biotin end-labeling was verified by gel-shift assays with NeutrAvidin (Pierce Chemicals) on 1% TBE agarose gels.
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| |
|
| Hybridization protocol |
The cDNA was hybridized to the MD4-9313 custom Affymetrix array in aqueous hybridization solution (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, 7.8 % DMSO and 3 nM prelabeled Affymetrix hybridization B2 oligo control probe mix) during a 16 h incubation at 45 °C in a GeneChip Hybridization Oven 320 rotating at 60 rpm. Washes and stains were carried out on a GeneChip Fluidics Station 450 (Affymetrix) following the ProkGE_WS2v3 Affymetrix protocol. Briefly, following two stringency washes the array was sequentially incubated with 10 µg/mL streptavidin (Pierce Chemical), 5 µg/mL biotinylated anti-streptavidin goat antibody (Vector Laboratories) and 0.1 mg/mL goat IgG (Sigma) and 10 µg/mL streptavidin-phycoerythrin conjugate (Mol. Probes) each for 10 min at 25 °C and given a final wash.
|
| Scan protocol |
The arrays were scanned with the GeneChip Scanner (Affymetrix) at a 2.5 µm resolution with excitation set for 570 nm.
|
| Description |
no additional information
|
| Data processing |
The probe set summaries were calculated from perfect match probe intensities in Affymetrix CEL files using robust multi-array average (RMA) analysis with quantile normalization (as implemented in the Bioconductor package affy).
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| |
|
| Submission date |
Jul 05, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Debbie Lindell |
| Organization name |
Technion - Israel Institute of Technology
|
| Department |
Department of Biology
|
| Street address |
Technion City
|
| City |
Haifa |
| ZIP/Postal code |
32000 |
| Country |
Israel |
| |
|
| Platform ID |
GPL5471 |
| Series (1) |
| GSE8382 |
Genome-Wide Expression Dynamics of a Marine Virus and its Host during Lytic Infection |
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