10 protocols
AccessionNameType
P-GSE41749-3
normalization data transformation protocol
Basecalls for both sequencing technologies performed using standard softwares supplied by either Roche for FLX sequencing or by Illumina for GAIIx sequencing. For FLX reads the barcodes at the 5’-end (C. albicans: 5’-CGAGAC-3’ and C. dubliniensis: 5’- CGTCGT-3’) and the remaining sequences from the B adapter at the 3’-end (5’-CTGAGACTGCCAAGGCACACAGGGGATAGG-3’) were trimmed. The processed reads were then blasted with BLASTN (ncbi-blast-2.2.23). Only unique and non-spliced reads with at least 90% identities over the whole length of the reads has passed the quality filter. For intron spanning reads we also performed BLAT alignments (BLAT Suite 0.34). GAIIx reads were mapped with their full length of 75 sequenced bases with SOAP2 (version 2.20) using a seed length of 40 bp allowing 5 mismatches with the exception for sample C.dubliniensis_GAIIx_YPS_blastospores-2.fastq where the first base of each sequence was trimmed resulting in 74 bp reads. As SOAP2 is not compatible with intron-spanning reads we performed another mapping with the remaining unmapped reads with TOPHAT (version 1.3.1). Genome_build: C. albicans: Assembly21 at CGD (latest update 2010-06-15, http://www.candidagenome.org/download/sequence/Assembly21); C. dubliniensis: Assembly CD36 at NCBI (latest update 2010-04-09, ftp://ftp.ncbi.nih.gov/genomes/Fungi/Candida_dubliniensis_CD36) Supplementary_files_format_and_content: BLASTN/BLAT output files were generated as table formats, then piped (shell script) into *.gff3 and finally into *.bedGraph. Standard SOAP output files were converted similarly. Standard TOPHAT output files were converted with BEDtools (bam-to-bed) into *.bed file format. Scores represent coverage of raw mapped reads.
P-GSE41749-4
growth protocol
Blastospore cells were grown in YPD supplemented with 10% fetal calf serum at 37°C for 4 hours while hyphal cells were induced in water supplemented with 10% fetal calf serum at 37°C for 4 hours.
P-GSE41749-2
nucleic acid library construction protocol
For the normalized cDNA libraries for FLXTitanium-sequencing, equal amounts of approximately 25 µg of total RNA per condition were pooled together per species. To get rid of genomic contaminants another purification step was performed using QIAGEN’s RNeasy Mini Plus Kit. From the pooled total RNA poly(A)+-RNA was prepared according to standard protocols (Ref.). First-strand cDNA synthesis was carried out with a N6 randomized primer. Then 454 adapters A (5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3’) and B (5’-CTGAGACTGCCAAGGCACACAGGGGATAGG-3’) were ligated to the 5' and 3' ends of the cDNAs, respectively, to obtain strand-specificity of the transcripts. Additionally, the C. albicans and the C. dubliniensis samples were barcoded at the 5’-end of the fragments with 5’-CGAGAC-3’ and 5’- CGTCGT-3’, respectively. The cDNAs were amplified with 16 cycles of PCR. Normalization was carried out by one cycle of denaturation and reassociation of the cDNA. Reassociated ds-cDNA was separated from the remaining ss-cDNA (normalized cDNA) by passing the mixture over a hydroxylapatite column. After hydroxylapatite chromatography, the ss-cDNA was amplified by 9 PCR cycles. For Titanium sequencing the cDNA in the size range of 500 – 700 bp was eluted from preparative agarose gels. Aliquots of the size fractionated cDNAs were analyzed by capillary electrophoresis with the Shimadzu MultiNA microchip system. The two normalized and barcoded cDNA libraries were pooled at equal amounts and were sequenced on a full flow cell with the Titanium chemistry resulting in about 500.000 reads per species. After 4h of incubation under the corresponding conditions, C. dubliniensis and C. albicans cells were harvested by centrifugation and immediately frozen with liquid nitrogen. Disruption was carried out using a Mixer Mill MM 200 (RETSCH) with a shaking frequency of 30/s. The resulting powder was resuspended in lysis buffer RLT (QIAGEN, Hilden, Germany) supplemented with 0.01% v/v of ß-mercaptoethanol. The extraction of total RNA was performed according to QIAGEN’s Mechanical Disruption Protocol for the isolation of total RNA from yeast using the RNeasy Midi Kit. After precipitation of the RNA by addition of 0.1 volume of 3M NaAc pH 5.3 and 2.5 volume of 100% EtOH, the concentration and integrity of total RNA was analyzed using the Agilent 2100 Bioanalyzer using the RNA Nano kit.
P-GSE41749-1
growth protocol
Blastospore cells were grown in YPD at 30°C for 4 hours while hyphal cells were induced in YPD supplemented with 10% fetal calf serum at 37°C for 4 hours.
P-GSE41749-10
normalization data transformation protocol
Basecalls were performed using standard software supplied by Illumina for HiSeq2000 sequencing. Reads generated with HiSeq2000 were mapped with TOPHAT (version 1.4.1) using default settings. Genome_build: C. albicans: Assembly21 at CGD (latest update 2010-06-15, http://www.candidagenome.org/download/sequence/Assembly21); C. dubliniensis: Assembly CD36 at NCBI (latest update 2010-04-09, ftp://ftp.ncbi.nih.gov/genomes/Fungi/Candida_dubliniensis_CD36) Supplementary_files_format_and_content: TOPHAT output files were converted with BEDtools (bam-to-bed) into *.bed file format. Non-junction reads were generated from *.bed files into *.bedGraph files by a customized python-script. Scores represent coverage of raw mapped reads
P-GSE41749-9
nucleic acid library construction protocol
After 4h of incubation under the corresponding conditions, C. dubliniensis and C. albicans cells were harvested by centrifugation and immediately frozen with liquid nitrogen. Disruption was carried out using a Mixer Mill MM 200 (RETSCH) with a shaking frequency of 30/s. The resulting powder was resuspended in lysis buffer RLT (QIAGEN, Hilden, Germany) supplemented with 0.01% v/v of ß-mercaptoethanol. The extraction of total RNA was performed according to QIAGEN’s Mechanical Disruption Protocol for the isolation of total RNA from yeast using the RNeasy Midi Kit. After precipitation of the RNA by addition of 0.1 volume of 3M NaAc pH 5.3 and 2.5 volume of 100% EtOH, the concentration and integrity of total RNA was analyzed using the Agilent 2100 Bioanalyzer using the RNA Nano kit. The cDNA libraries for HiSeq2000-sequencing were performed according Illumina’s TruSeq RNA Sample Prep Kit v2 protocol. The sequencing run was performed on single-read flow cell with 50bp reads and resulted in approximately 10-15 mio. reads per sample.
P-GSE41749-8
growth protocol
C. albicans_YPD-1, C. albicans_YPD-2, C. albicans_YPD-3 samples were grown in YPD at 30°C for 4 hours. C. albicans_YPS-3 and C. dubliniensis_YPS-3 samples were grown in YPD supplemented with 10% fetal calf serum at 37°C for 4 hours. C. dubliniensis_WS-3 sample was grown in water supplemented with 10% fetal calf serum at 37°C for 4 hours.
P-GSE41749-6
nucleic acid library construction protocol
The cDNA libraries for GAIIx-sequencing were performed according Illumina’s mRNA-Seq Sample Prep Kit protocol. Each of the six samples was loaded on one lane. Thus, the sequencing run was performed on a fully loaded flow cell with single-end 76bp reads and resulted in approximately 30 mio. reads per sample. After 4h of incubation under the corresponding conditions, C. dubliniensis and C. albicans cells were harvested by centrifugation and immediately frozen with liquid nitrogen. Disruption was carried out using a Mixer Mill MM 200 (RETSCH) with a shaking frequency of 30/s. The resulting powder was resuspended in lysis buffer RLT (QIAGEN, Hilden, Germany) supplemented with 0.01% v/v of ß-mercaptoethanol. The extraction of total RNA was performed according to QIAGEN’s Mechanical Disruption Protocol for the isolation of total RNA from yeast using the RNeasy Midi Kit. After precipitation of the RNA by addition of 0.1 volume of 3M NaAc pH 5.3 and 2.5 volume of 100% EtOH, the concentration and integrity of total RNA was analyzed using the Agilent 2100 Bioanalyzer using the RNA Nano kit.
P-GSE41749-7
growth protocol
Cells were grown in water supplemented with 10% fetal calf serum at 37°C for 4 hours.
P-GSE41749-5
growth protocol
Cells were grown in YPD supplemented with 10% fetal calf serum at 37°C for 4 hours.