|
| Status |
Public on May 22, 2013 |
| Title |
H4-depletion_300min [transcript profile] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
UKY403 after 5 hours of histone H4 depletion
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain background: UKY403, MATa ade2-101 his3-A200 leu2-3, 112 lys-801 trpl-A901 ura3-52 GAL+ thr tyr arg4-1 Ah4-J[HIS3+] Ah4-2[LEU2+ ]/pUK421 (CEN TRPI+ GAL-H4-2 +)
|
| Growth protocol |
For the histone depletion time course, the UKY403 strain harboring a deletion of both Histone H4 genes (HHF1, HHF2), a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2) and a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2), was grown in YP with 2% galactose until mid log phase. Cells were then collected by centrifugation and transferred to YPD for 0, 1⁄4,1, 3, 5, and 6 hrs. After each time point, samples were taken for nucleosome and expression analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from yeast cells by vortexing with glass beads in the presence of hot acid phenol and cleaned by phenol:chlorophorm extraction followed by ethanol precipitation. After DNaseI (GE Healthcare) treatment to remove residual genomic DNA, 25 ug of total RNA was incubated with random hexamers at 70C for 10 min, incubated at 15C for 30 min and reverse transcribed to single-stranded cDNA for 2 hours at 42C with Superscript II (Invitrogen) in the presence of 6 μg/ml Actinomycin D to prevent antisense artifacts. RNA was subsequently removed by hydrolysis and cDNA was cleaned up and concentrated on MinElute PCR purification columns (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
cDNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37ｰC until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP (Enzo Life Sciences) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
|
| |
|
| Channel 2 |
| Source name |
total RNA from matched wild type control
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain background: UKY403, MATa ade2-101 his3-A200 leu2-3, 112 lys-801 trpl-A901 ura3-52 GAL+ thr tyr arg4-1 Ah4-J[HIS3+] Ah4-2[LEU2+ ]/pUK421 (CEN TRPI+ GAL-H4-2 +)
genotype/variation: wild type
|
| Growth protocol |
For the histone depletion time course, the UKY403 strain harboring a deletion of both Histone H4 genes (HHF1, HHF2), a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2) and a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2), was grown in YP with 2% galactose until mid log phase. Cells were then collected by centrifugation and transferred to YPD for 0, 1⁄4,1, 3, 5, and 6 hrs. After each time point, samples were taken for nucleosome and expression analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from yeast cells by vortexing with glass beads in the presence of hot acid phenol and cleaned by phenol:chlorophorm extraction followed by ethanol precipitation. After DNaseI (GE Healthcare) treatment to remove residual genomic DNA, 25 ug of total RNA was incubated with random hexamers at 70C for 10 min, incubated at 15C for 30 min and reverse transcribed to single-stranded cDNA for 2 hours at 42C with Superscript II (Invitrogen) in the presence of 6 μg/ml Actinomycin D to prevent antisense artifacts. RNA was subsequently removed by hydrolysis and cDNA was cleaned up and concentrated on MinElute PCR purification columns (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
cDNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37ｰC until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP (Enzo Life Sciences) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
|
| |
|
| |
| Hybridization protocol |
6 μg of biotinylated cDNA was hybridized at 45C for 16 hours, and then washed and stained according to Affymetrix protocol EukGE-WS2v4_450 in an Affymetrix Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned in an Affymetrix 7G scanner.
|
| Description |
ctrl CEL file: MHY5.CEL
H4-depletion_300min
Expression profile after 5 hours of histone H4 depletion compared to wild-type control
|
| Data processing |
Raw data files were normalized with Affymetrix Tiling Analysis Software (TAS) v1.1. Treatment and control samples were quantile normalized in pairs using only perfect match probes with a bandwidth of 20. The files ending in 'forward.bar' and 'reverse.bar' correspond to normalized signals from the forward and reverse strand, respectively.
|
| |
|
| Submission date |
Mar 05, 2013 |
| Last update date |
May 22, 2013 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
[email protected]
|
| Organization name |
Mount Sinai School of Medicine
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel Lab
|
| Street address |
One Gustave L. Levy Place, Box 1498
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL7070 |
| Series (2) |
| GSE44876 |
A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription [transcript profiling] |
| GSE44879 |
A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription |
|
