|
| Status |
Public on Dec 01, 2014 |
| Title |
ChIP_Rpb3_IP |
| Sample type |
genomic |
| |
|
| Source name |
Yeast cells grown in YPD to exponential phase
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: WT strain: BY4741 (SC288 background)
antibody: Anti-POLR2C antibody [1Y26 (1Y27)]-ChIP Grade
antibody manufacturer: Abcam
antibody catalog number: ab81859
|
| Treatment protocol |
For BioGRO samples, cells were collected by centrifugation and frozen in liquid nitrogen. Frozen pellets were transferred immediately to -20 °C. After at least 3 h, cells were thawed on ice and permeabilized with 10 mL of a 0.5% sarkosyl solution. Once permeabilized, cells were treated with RNase A (Roche). RNase trimming was achieved by incubating the cells with 32 µL of RNase A (10 mg/mL) dissolved in 3.2 mL of Sarkosyl 0.5% in agitation for 10 min at 30 °C. In order to remove the RNase, cells were washed three times with 50 mL 0.5% sarkosyl, and then transferred to an eppendorf tube. Cells were resuspended in 115 µL of water plus 5 µL of RNase OUT (Invitrogen) to protect the integrity of nascent RNAs from any residual RNase that might be present. Run-on reactions where performed as described (Jordán-Pla et al., 2014). For ChIP samples we followed the Affymetrix® Chromatin Immunoprecipitation Assay Protocol (PN 702238).
|
| Growth protocol |
All yeast strains where grown in standard conditions (YPD medium, 30 ºC). For each sample we used an aliquot of 100 mL cells grown to mid exponetial phase (DO600 = 0.55).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For BioGRO samples, RNA extraction was done using the “MasterPure Yeast RNA Purification Kit” (Epicentre), following manufacturer’s instructions. Once extracted, genomic DNA was removed digesting with 2 µL of RNase-free DNase I (Roche) for 30 min at 37 °C. The purified RNA was resuspended in 32 µL of water and spectrophotometrically quantified. For ChIP samples, we followed the Affymetrix® Chromatin Immunoprecipitation Assay Protocol (PN 702238).
|
| Label |
Biotin
|
| Label protocol |
For BioGRO samples, labeling of nascent RNAs was achieved by incorporating Bio-11-UTP (Ambion) to the nascent chains in vivo by run-on. For ChIP samples, we followed Affymetrix® Chromatin Immunoprecipitation Assay Protocol (PN 702238).
|
| |
|
| Hybridization protocol |
For BioGRO samples, we followed the instructions of the GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual (part # 701880), starting from Chapter 5: Hybridization, using the GeneChip® Hybridization, Wash and Stain Kit. For ChIP samples, we followed the Affymetrix® Chromatin Immunoprecipitation Assay Protocol (PN 702238).
|
| Scan protocol |
For BioGRO samples, we followed the instructions of the GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual (part # 701880), scanning the array as described in Chapter 7: Scanning. For ChIP samples, we followed the Affymetrix® Chromatin Immunoprecipitation Assay Protocol (PN 702238), scanning the arrays as described in Chapter 5: Scan.
|
| Data processing |
Raw .CEL images were processed with Tiling Analysis Software (TAS, Affymetrix) with the signal detection parameters set by default. BioGRO samples where normalized against a reference genomic DNA hybridized on the same Affymetrix tiling arrays (ArrayExpress E-TABM-590). The ChIP (IP) sample was normalized against its respective Input sample (Input).
The resulting files were obtained with Tiling Analysis Software (TAS, Affymetrix) with the signal detection parameters set by default. BioGRO samples where normalized against a reference genomic DNA hybridized on the same Affymetrix tiling arrays (ArrayExpress E-TABM-590). The ChIP (IP) sample was normalized against its respective Input sample (Input).
|
| |
|
| Submission date |
Jun 26, 2014 |
| Last update date |
Dec 01, 2014 |
| Contact name |
Jose E. Perez-Ortin |
| E-mail(s) |
jose.e.perez@uv.es
|
| Phone |
34 963 543467
|
| Organization name |
Universitat de Valencia
|
| Department |
Bioquimica y Biologia Molecular
|
| Lab |
Yeast Functional Genomics (GFL)
|
| Street address |
Dr. Moliner 50
|
| City |
Burjassot |
| State/province |
Valencia |
| ZIP/Postal code |
E46100 |
| Country |
Spain |
| |
|
| Platform ID |
GPL18871 |
| Series (1) |
| GSE58859 |
Chromatin-dependent regulation of the RNA polymerases II and III activity throughout the transcription cycle |
|
