 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 30, 2011 |
| Title |
5EL4_YPD_30_O1_UPC2KO |
| Sample type |
SRA |
| |
|
| Source name |
Total RNA from Candida parapsilosis
|
| Organism |
Candida parapsilosis |
| Characteristics |
strain: CDupc5
medium: YPD
temperature: 30 degree Celsius
oxygen concentration: 1%
|
| Growth protocol |
Cells were grown in either YPD (1% Yeast Extract, 2% Peptone, 2% Glucose; FormediumTM), YPGlycerol (YPD with 2% glycerol instead of 2% glucose) or BMW (1.5% Yeast Extract, 1% Peptone, 2% Glucose, 0.2% SC Amino Acid mix, 0.01% Adenine, 0.01% Tryptophan, 0.01% Uracil); Cells were grown at 30°C and 21% O2 unless otherwise noted. Where indicated, cells were grown in 1% oxygen, 99% nitrogen in an InVivo2 400 hypoxic chamber and in two libraries, the temperature used was either 30 or 37 degree Celsius. In some strains the transcription factor UPC2 was deleted.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
mRNA purified from total RNA using oligo dT Dynabeads (Invitrogen) was fragmented to an average size of 200 nucleotides by a 5 minute heat treatment (70°C) with a buffered zinc solution (Fragmentation Reagent, Ambion). Fragmentation of mRNA was stopped using an EDTA based Stop buffer (Ambion). Fragmented mRNA was incubated with 3 μg Random Hexamer Primers at 65°C for 5 min. First strand cDNA synthesis was carried out using 1 x First Strand Buffer (Invitrogen), 10 mM DTT, 500 μM dNTP mix (Invitrogen), 20 Units RNaseOUT and 200 units SuperScriptTM II Reverse Transcriptase (Invitrogen). For strand-specific library generation, unincorporated dNTPs were subsequently removed using G-50 Micro Columns (GE Healthcare). Second strand cDNA was generated using 1x Second Strand Buffer, 300 μM dNTP mix, 2 units RNaseH, 50 units DNA Polymerase I (NEB), while a 300 μM dUTP mix was used instead of a dNTP mix for the generation of a strand specific library. This material was used for library preparation. For strand-specific libraries, DNA fragments were blunted in an End Repair reaction using T4 DNA Polymerase, Klenow DNA Polymerase and T4 Polynucleotide Kinase, after which a single ‘A’ base was added to the 3’ end using dATP and Klenow Exo Fragment. Illumina adapters were ligated to the ends of the DNA fragments, allowing for the subsequent hybridization to a flow cell. Fragments of approximately 300 base pairs were purified from a 2% Agarose Gel. For the strand-specific libraries, the second strand containing uridine was removed by treatment with 1 unit Uracil N-glycosylase (UNG) in TE Buffer at 37°C for 15 min. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR. All libraries were quantified using a Qubit® Fluorometer (Invitrogen) and assessed on a 2% agarose gel. Amplified libraries were loaded on a flow cell. Sequencing was carried out in-house by running at least 36 cycles on an Illumina Genome Analyzer IIx according to manufacturer’s instructions, resulting in read lengths of approximately 42 bases. In addition, long reads (76 bases) were generated from two libraries by GATC Biotech AG using an Illumina Genome Analyzer IIx.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
upc2 deletion
|
| Data processing |
Alignment: Sequence reads were obtained using the Illumina Genome Analyzer Pipeline. Reads were mapped to the Candida parapsilosis genome, All reads mapping only in one location with two or fewer mismatches were retained
|
| |
|
| Submission date |
Oct 10, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Geraldine Butler |
| E-mail(s) |
geraldine.butler@ucd.ie
|
| Organization name |
University Colege Dublin Conway Institute
|
| Department |
School of Biomolecular and Biomedical Science
|
| Lab |
Butler lab
|
| Street address |
Belfield
|
| City |
Dublin |
| ZIP/Postal code |
D4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL14718 |
| Series (2) |
| GSE32714 |
Transcriptional landscape of Candida parapsilosis |
| GSE32716 |
Transcriptional landscape of the pathogenic yeast Candida parapsilosis |
|
| Relations |
| SRA |
SRX100815 |
| BioSample |
SAMN00738673 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM812972_5EL4_YPD_30_O1_UPC2KO.sam.gz |
443.5 Mb |
(ftp)(http) |
SAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
