|
| Status |
Public on Aug 25, 2016 |
| Title |
mRNA levels 32min after release from G1 with EdU dye flip, rep2 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
cDNA mid S-phase +EdU
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: MATa ade2-1 trp1-1 can1-1000 leu2-3,112 his3-11,15 GAL psi+ RAD5+ ura3::URA3/GPD-TK(7x) AUR1c::ADH-hENT1 bar1D::KANR
growth phase: mid S-phase
treatment: EdU
|
| Treatment protocol |
50ml aliquots were flash frozen in liquid nitrogen before and 32 and 40min after release from G1 arrest.
|
| Growth protocol |
Cells were grown overnight at 30°C in Synthetic Complete- URA + Dextrose media to OD 0.3. After 3.75hrs of incubation at 30°C with alpha factor (0.25μg/ml), cells were pelleted and transferred into preheated and premixed SCD-URA+ or - 10μM EdU (Carbosynth) as indicated, with freshly added 20μg/ml pronase (Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Genomic DNA was isolated from G1 arrested flash frozen cell pellets with Phenol/Chloroform, and sonicated with the Bioruptor Pico cup sonicator (200µl at 200ng/ µl, 30”ON 30”OFF at 4°C). Total RNA was isolated from frozen PV1 cell pellets with Trizol. Frozen cell pellets were resuspended directly in Trizol and bead beated in the Bullet Blender (Next Advance). RNA was then purified and DNaseI treated with the RNeasy Column purification kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
We used ~30 µg of total RNA for each expression array. RNA was reverse transcribed using oligodTs (0.15µg/µl each) as primers. Reactions (0.5mM dNTP (N=A,G,C), 0.2mMdTTP and 0.3mM amino-allyl dUTP (SIGMA), 6µg/ml Actinomycine D (SIGMA), 10mMDTT, 1XFS buffer and 10U/µl Superscript III (Life Technologies)) were incubated at 50°C for 2hrs. RNA was then degraded with NaOH at 65°C (10µl 1N NaOH and 10µl 0.5M EDTA into 30µl reactions), the solution was neutralized with HEPES pH=7.5 (25µl 1M stock) and the buffer was exchanged for water in Amicon30 centricon spin columns. The resulting cDNA was dye-coupled with Cy5 or Cy3 NHS-esters and purified as described previously (Liu et al., 2005). Genomic DNA labeling: 2ug (quantified in the Qubit fluorimeter) PV1 G1 genomic DNA in Klenow NEB buffer, 0.3 µg/µl random hexamers, 0.12 mM dNTP (N=A,G,T),0.06mM dCTP and 0.06mM Cy5 or Cy3 conjugated dCTP (GE healthcare), and 1U/µl Klenow enzyme (NEB); incubated 2hrs at 37°C and cleaned up in Amicon-30 centricon spin columns.
|
| |
|
| Channel 2 |
| Source name |
G1 genomic DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: MATa ade2-1 trp1-1 can1-1000 leu2-3,112 his3-11,15 GAL psi+ RAD5+ ura3::URA3/GPD-TK(7x) AUR1c::ADH-hENT1 bar1D::KANR
growth phase: G1
|
| Treatment protocol |
50ml aliquots were flash frozen in liquid nitrogen before and 32 and 40min after release from G1 arrest.
|
| Growth protocol |
Cells were grown overnight at 30°C in Synthetic Complete- URA + Dextrose media to OD 0.3. After 3.75hrs of incubation at 30°C with alpha factor (0.25μg/ml), cells were pelleted and transferred into preheated and premixed SCD-URA+ or - 10μM EdU (Carbosynth) as indicated, with freshly added 20μg/ml pronase (Sigma).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from G1 arrested flash frozen cell pellets with Phenol/Chloroform, and sonicated with the Bioruptor Pico cup sonicator (200µl at 200ng/ µl, 30”ON 30”OFF at 4°C). Total RNA was isolated from frozen PV1 cell pellets with Trizol. Frozen cell pellets were resuspended directly in Trizol and bead beated in the Bullet Blender (Next Advance). RNA was then purified and DNaseI treated with the RNeasy Column purification kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
We used ~30 µg of total RNA for each expression array. RNA was reverse transcribed using oligodTs (0.15µg/µl each) as primers. Reactions (0.5mM dNTP (N=A,G,C), 0.2mMdTTP and 0.3mM amino-allyl dUTP (SIGMA), 6µg/ml Actinomycine D (SIGMA), 10mMDTT, 1XFS buffer and 10U/µl Superscript III (Life Technologies)) were incubated at 50°C for 2hrs. RNA was then degraded with NaOH at 65°C (10µl 1N NaOH and 10µl 0.5M EDTA into 30µl reactions), the solution was neutralized with HEPES pH=7.5 (25µl 1M stock) and the buffer was exchanged for water in Amicon30 centricon spin columns. The resulting cDNA was dye-coupled with Cy5 or Cy3 NHS-esters and purified as described previously (Liu et al., 2005). Genomic DNA labeling: 2ug (quantified in the Qubit fluorimeter) PV1 G1 genomic DNA in Klenow NEB buffer, 0.3 µg/µl random hexamers, 0.12 mM dNTP (N=A,G,T),0.06mM dCTP and 0.06mM Cy5 or Cy3 conjugated dCTP (GE healthcare), and 1U/µl Klenow enzyme (NEB); incubated 2hrs at 37°C and cleaned up in Amicon-30 centricon spin columns.
|
| |
|
| |
| Hybridization protocol |
Labeled probes (a mixture of Cy3 labeled cDNA/genomic DNA and Cy5 labeled genomic DNA/cDNA) were hybridized onto an Agilent yeast 8x15 gene expression array.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with an InnoScan 710 MicroArray scanner (Innopsys) at 5 micron resolution and processed with the Mapix software.
|
| Description |
cDNA_CY3/gDNA_Cy5_32minEdU replicate 2
|
| Data processing |
After background correction and removal of flagged values, data was block normalized by dilation and log base 2 ratios (Cy3/Cy5) were calculated to produce the values given in the data table.
|
| |
|
| Submission date |
Jul 21, 2016 |
| Last update date |
Aug 25, 2016 |
| Contact name |
Marta Radman-Livaja |
| E-mail(s) |
mrl5374@gmail.com
|
| Phone |
+33434359667
|
| Organization name |
CNRS
|
| Department |
IGMM
|
| Street address |
1919 route de Mende
|
| City |
Montpellier |
| ZIP/Postal code |
34293 |
| Country |
France |
| |
|
| Platform ID |
GPL9825 |
| Series (1) |
| GSE79384 |
Dynamics of nucleosome positioning maturation following genomic replication |
|
