|
| Status |
Public on Nov 20, 2018 |
| Title |
CRZ2_ChIP_Upper array |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Candida albicans untagged strains, treated with 40 µg/ml doxycycline for 4h in YPD medium at 30ºC, immunoprecipitated DNA
|
| Organism |
Candida albicans |
| Characteristics |
genotype/variation: PTET-CRZ2
treatment: doxycycline
chip antibody: mouse IgG
|
| Growth protocol |
Cells were grown overnight in 2 ml YPD at 30°C, diluted to an OD600 nm of 0.3 in YPD medium supplemented with 40 µg/ml doxycycline and re-grown for 4h at 30ºC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
PTET-TAP-CRZ2 (Tagged) and PTET-CRZ2 (untagged control) were grown overnight in 2 ml YPD at 30°C, diluted to an OD600 nm of 0.3 in YPD medium supplemented with 40 g/ml doxycycline and grown during 4 hours at 30°C. Cultures were treated with 1% formaldehyde (cross-linking) and snap-frozen in liquid nitrogen. Total cell extracts were prepared by bead beating using a FastPrep-24 instrument (MP Biomedicals) with 10 runs during 40 seconds each at 5.5 m/sec and 1 minute on ice in between. Soluble chromatin fragments were prepared by sonicating the extracts 6 times during 20 sec at power 8 (knob position) for an output signal amplitude of 15 (Microns, Peak to Peak) using a probe sonicator (MSE), yielding a majority of ~200-500 bp DNA fragments. Immunoprecipitation was conducted overnight at 4ºC with 500 µl of clarified sonicated extracts and 40 µl of IgG-coated magnetic beads (Dynabeads Pan mouse IgG, Invitrogen, cat# 11041, lot# 120572900), previously prehybed overnight with PBS-0.1% BSA at 4ºC. The concentration of the purified immunoprecipitated (IP) DNA was ranging between 31 pg/µl and 147 pg/µl in 50 µl TE (10 mM Tris [pH 8.0], 1 mM EDTA).
|
| Label |
Cy3
|
| Label protocol |
The IP DNA (40 µl out of 50 µl) fragments were blunted with T4 DNA polymerase and ligated to unidirectional linkers. The DNA was amplified by ligation-mediated PCR in the presence of aminoallyl-modified dUTP. Labeling was carried out post-PCR using monoreactive Cy dye N-hydroxysuccinimide esters (Cy5/Cy3 monoreactive dye packs; Amersham Biosciences) that react specifically with the aminoallyl-modified dUTP [5-(3-aminoallyl)-2′ deoxyuridine-5′-triphosphate; Sigma-Aldrich]. Labeled IP DNA from the tagged strain (Cy5) and the untagged control strain (Cy3) were mixed and hybridized to C. albicans tiling arrays.
|
| |
|
| Channel 2 |
| Source name |
Candida albicans strains expressing a TAP-tagged version of the CRZ2 gene placed under the control of a doxycycline-inducible promoter, treated with 40 µg/ml doxycycline for 4h in YPD medium at 30ºC, immunoprecipitated DNA
|
| Organism |
Candida albicans |
| Characteristics |
genotype/variation: PTET-TAP-CRZ2
treatment: doxycycline
chip antibody: mouse IgG
|
| Growth protocol |
Cells were grown overnight in 2 ml YPD at 30°C, diluted to an OD600 nm of 0.3 in YPD medium supplemented with 40 µg/ml doxycycline and re-grown for 4h at 30ºC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
PTET-TAP-CRZ2 (Tagged) and PTET-CRZ2 (untagged control) were grown overnight in 2 ml YPD at 30°C, diluted to an OD600 nm of 0.3 in YPD medium supplemented with 40 g/ml doxycycline and grown during 4 hours at 30°C. Cultures were treated with 1% formaldehyde (cross-linking) and snap-frozen in liquid nitrogen. Total cell extracts were prepared by bead beating using a FastPrep-24 instrument (MP Biomedicals) with 10 runs during 40 seconds each at 5.5 m/sec and 1 minute on ice in between. Soluble chromatin fragments were prepared by sonicating the extracts 6 times during 20 sec at power 8 (knob position) for an output signal amplitude of 15 (Microns, Peak to Peak) using a probe sonicator (MSE), yielding a majority of ~200-500 bp DNA fragments. Immunoprecipitation was conducted overnight at 4ºC with 500 µl of clarified sonicated extracts and 40 µl of IgG-coated magnetic beads (Dynabeads Pan mouse IgG, Invitrogen, cat# 11041, lot# 120572900), previously prehybed overnight with PBS-0.1% BSA at 4ºC. The concentration of the purified immunoprecipitated (IP) DNA was ranging between 31 pg/µl and 147 pg/µl in 50 µl TE (10 mM Tris [pH 8.0], 1 mM EDTA).
|
| Label |
Cy5
|
| Label protocol |
The IP DNA (40 µl out of 50 µl) fragments were blunted with T4 DNA polymerase and ligated to unidirectional linkers. The DNA was amplified by ligation-mediated PCR in the presence of aminoallyl-modified dUTP. Labeling was carried out post-PCR using monoreactive Cy dye N-hydroxysuccinimide esters (Cy5/Cy3 monoreactive dye packs; Amersham Biosciences) that react specifically with the aminoallyl-modified dUTP [5-(3-aminoallyl)-2′ deoxyuridine-5′-triphosphate; Sigma-Aldrich]. Labeled IP DNA from the tagged strain (Cy5) and the untagged control strain (Cy3) were mixed and hybridized to C. albicans tiling arrays.
|
| |
|
| |
| Hybridization protocol |
The labeled DNA was speed-vaced and the pellets were resuspended in 104 µl of H2O. 26 µl of Agilent 10X blocking agent and 130 µl of Agilent 2X Hybridization Buffer were added for a total of 260 µl hybridization mix. Samples were heated at 95ºC for 3 minutes and incubated immediately at 37ºC during 30 min. 245 µl were dispensed onto each of the 2x400k Agilent gaskets. The hybridization chambers were assembled and rotated overnight at 65ºC as per the manufacturer's protocol.
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol.
|
| Description |
Sample 1
Background strain is BWP17 carrying a homozygous deletion of CRZ2 and expressing TAP-tagged or untagged CRZ2 alleles under the control of the tetracycline derivative-inducible promoter.
|
| Data processing |
Data were quantile normalized using CisGenome version 1.0 (Hongkai et al. 2005 Bioinformatics, 21: 3629-3636).
|
| |
|
| Submission date |
Mar 24, 2015 |
| Last update date |
Nov 20, 2018 |
| Contact name |
Sadri ZNAIDI |
| E-mail(s) |
sadri.znaidi@gmail.com
|
| Organization name |
Institut Pasteur
|
| Department |
Mycology Department
|
| Lab |
Fungal Biology and Pathogenicity
|
| Street address |
25 rue du Docteur Roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
| |
|
| Platform ID |
GPL17892 |
| Series (2) |
| GSE67233 |
Genome-wide location of Candida albicans transcription factor Crz2p |
| GSE67235 |
Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut |
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