| attributes |
| Alias |
E-MTAB-4468:Carbon limitation 1 |
| Broker name |
ArrayExpress |
| Description |
Protocols: Samples of steady-state R. pomeroyi DSS-3 cells (45 l) were collected from chemostats after five volume exchanges. An RNA stabilization solution (95% ethanol 5% phenol) was added to constitute 10% of the total volume and cells were centrifuged. Pellets were stored frozen at -80oC until processing. R. pomeroyi DSS-3 cells used for transcriptome sequencing and RT-qPCR analysis were grown in carbon- and nitrogen-limited chemostats. Three replicates were run in each condition (carbon limitation, 1 mmol l-1 glucose and 2.8 mmol l-1 NH4Cl; nitrogen limitation, 4.5 mmol l-1 glucose and 0.26 mmol l-1 NH4Cl). A continuous culturing approach was used in order to study growth under chronic nutrient limitation rather than the physiologically distinct process of nutrient starvation and shift to stationary phase. Additional details of the chemostat design are found in Chan et al. (2012). For RNA extraction, pellets were thawed and extracted using TriReagent (Molecular Research Center, Cincinnati, OH, USA). Purified RNA was depleted of rRNA with the MicrobeExpress Kit (Ambion/Applied Biosystems, Austin, TX) and the mRNA-enriched RNA was subsequently amplified using a strand-specific protocol (MessageAmpII-Bacteria Kit; Ambion/Applied Biosystems). Using the SOLiD Whole Transcriptome Analysis Kit (Applied Biosystems), 5 g of amplified mRNA from six samples (triplicates from both the C- and N-limitation treatments) were fragmented with RNaseIII and purified and concentrated with the RiboMinus kit (Invitogen). mRNA was examined for fragment length (Agilent 2100 Bioanalyzer) to ensure that the majority were in the 100-200 nt range. All procedures for adaptor ligation and cDNA synthesis were conducted according to the SOLiD protocol. Resultant cDNA was purified and concentrated using the MinElute PCR Purification Kit (Invitrogen), heat-denatured at 95oC, run on a Novex 6% TBE-Urea Gel (Invitrogen) under denaturing conditions with a 50 bp DNA ladder, and stained with SYBR Gold nucleic acid stain. Gel bands containing 100-200 nt cDNA (insert size 50-150 nt) were used for PCR amplification of cDNA using AmpliTaq DNA Polymerase. PCR was carried out with a 5 SOLiD primer and a barcoded 3 primer (using a unique barcode for each sample) for 16 cycles. Amplified cDNA was purified and concentrated using PureLink PCR Micro Kit (Invitrogen). |
| ENA checklist |
ERC000011 |
| INSDC center name |
DOE Joint Genome Institute |
| INSDC first public |
2016-02-27T17:12:41Z |
| INSDC last update |
2016-02-17T16:25:21Z |
| INSDC status |
public |
| SRA accession |
ERS1063435 |
| Sample Name |
ERS1063435 |
| Title |
Carbon limitation 1 |
| ammonium concentration |
0.0028 |
| glucose concentration |
0.001 |
| growth condition |
carbon limitation |
| organism |
Ruegeria pomeroyi DSS-3 |
|