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Sample GSM1093264 Query DataSets for GSM1093264
Status Public on May 22, 2013
Title SIR2_del [transcript profile]
Sample type RNA
 
Channel 1
Source name SIR2 deletion strain
Organism Saccharomyces cerevisiae
Characteristics strain background: BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
genotype/variation: SIR2 deletion strain
Growth protocol For deletion strains, 5 mL cultures were grown overnight at 30 °C in YPD. Cells were diluted to OD600 0.2/mL in 400 mL of YPD media the next morning, and grown to mid-log phase (OD600 0.8-1.0/ml) in 1L flasks at 30 °C while shaking, at which point RNA and nucleosomal DNA were isolated.
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: BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
genotype/variation: wild type
Growth protocol For deletion strains, 5 mL cultures were grown overnight at 30 °C in YPD. Cells were diluted to OD600 0.2/mL in 400 mL of YPD media the next morning, and grown to mid-log phase (OD600 0.8-1.0/ml) in 1L flasks at 30 °C while shaking, at which point RNA and nucleosomal DNA were isolated.
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; b13_WT1_cDNA.CEL
SIR2_del
Expression profile for SIR2 deletion strain 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) harm.vanbakel@mssm.edu
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

Supplementary file Size Download File type/resource
GSM1093264_SIR2_del_F.bar.gz 8.2 Mb (ftp)(http) BAR
GSM1093264_SIR2_del_R.bar.gz 8.2 Mb (ftp)(http) BAR
GSM1093264_b13_SIR2_cDNA_del.CEL.gz 28.4 Mb (ftp)(http) CEL
Processed data provided as supplementary file

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