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Sample GSM239950 Query DataSets for GSM239950
Status Public on Dec 07, 2007
Title pull-downC_11, ZT6
Sample type RNA
 
Source name Homer1a expressing cells, control
Organism Mus musculus
Characteristics mRNAs pull-down, control
Treatment protocol At light onset, animals were either sleep deprived by gentle handling or left undisturbed for 6 hrs.
Growth protocol Experiments were performed on 12 weeks-old (+/- 1 week) male mice. Animals were sleep deprived at light onset or left undisturbed for 6 hrs and randomly sacrificed by cervical dislocation.
Extracted molecule polyA RNA
Extraction protocol Mice were sacrificed by cervical dislocation. Total RNA: Whole brain total RNA was extracted and treated with DNase using the RNeasy Lipid Tissue Kit (Qiagen). PolyA RNA: the mRNA-tagging immunoprecipitation was modified from formerly published protocols. (P. J. Roy et al., 2002) (L. O. F. Penalva et al., 2004). Mice were anesthetized with pentobarbital (75mg/kg), and subsequently perfused with 20ml PBS-heparin (2U/ml), 20 ml PBS-formaldehyde 1%, 15ml gylcine 150mM, 20ml Tris-HCl 20mM, and 15ml NaCl 150mM. Brains were removed and homogenized at maximum speed on ice in 3ml lysis buffer (Hepes KOH pH7.3 50mM, EDTA 1mM, KCl 140mM, glycerol 10%, NP40 0.5%, dithiothreitol 1mM, vanadyl ribonucleoside complex 2mM, heparin 1000U/ml, 1 complete mini protease inhibitor cocktail tablet/50ml). Large debris were removed by centrifugation at 18’000g for 20 minutes at 4°C. The supernatants were incubated with 400μl of anti-FLAG M2 affinity gel beads (Sigma) and 80U Rnasine overnight at 4°C. After centrifugation, the pull-down supernatants were recovered for total RNA extraction. The beads were then washed three times with 7ml of lysis buffer, twice with 7ml of wash buffer (50mM Hepes-KOH pH7.3, 1mM EDTA, 1M KCl, 10% glycerol, 0.5%NP-40, 1mM DTT) and once with 7ml of TE (10mM Tris-HCl pH 7.5, 0.5mM EDTA). The RNA-protein crosslink was reversed by incubating the beads with 3ml of elution buffer for 6 hours at 65°C (50mM Tris-HCl pH7.5, 10mM EDTA, 1% SDS, 2mM ribonucleoside vanadyl complex). Proteins were then digested with proteinase K for 45 minutes at 50°C. Finally mRNAs were isolated from proteins and DNA by a double phenol-chloroform extraction followed by isopropanol precipitation. The mRNA pellets were rinsed twice with 0.5ml 70% ethanol and dissolved in 20μl RNase free water.
Label biotin
Label protocol The quality of the mRNAs was investigated on Agilent 2100 bioanalyzer chips. 10ng of immunoprecipitated mRNA was amplified with the WT-Ovation™ Pico RNA Amplification System (catalog #3300-12; Manuel version 1,0).Using the FL-Ovation cDNA Biotin Module V2 (catalog #4200-12) 5μg of cDNA was used to generate labeled targets for hybridization to Affymetrix GeneChip® arrays.
 
Hybridization protocol Affymetrix HG-U133 plus 2.0 microarrays (Affymetrix) were hybridized to 5 ug of labeled, amplified material and then, washed, stained and scanned according to the protocol described in Affymetrix GeneChip® Expression analysis manual. The fluidics protocol used was GE_WS2v4_450 as recommended by NuGen and not the GE_WS2v5_450 used for the Affymetrix amplification.
Scan protocol The GeneChip Scanner 3000 (Affymetrix) was used to collect signals after excitation at 570 nm.
Description none
Data processing Normalized expression signals were calculated from Affymetrix CEL files using the expresso function from Bioconductor affy package. This normalization follows the same procedure as for RMA, but replaces a quantiles normalization by a scaling normalization (Gautier et al.).
 
Submission date Oct 26, 2007
Last update date Aug 28, 2018
Contact name Mehdi Tafti
E-mail(s) mehdi.tafti@unil.ch
Phone +41216923971
Organization name University of Lausanne
Department CIG
Street address Genopode
City Lausanne
ZIP/Postal code 1015
Country Switzerland
 
Platform ID GPL1261
Series (2)
GSE9443 Gene expression in brain Homer1a-expressing cells after sleep deprivation
GSE9444 Sleep deprivation and the brain
Relations
Reanalyzed by GSE119085

Data table header descriptions
ID_REF
VALUE RMA; log2 normalized expression signal

Data table
ID_REF VALUE
1415670_at 8.422173463
1415671_at 6.517228828
1415672_at 8.644192638
1415673_at 6.949806585
1415674_a_at 6.14252322
1415675_at 7.705324531
1415676_a_at 7.113994579
1415677_at 5.967956387
1415678_at 8.22351217
1415679_at 6.953394577
1415680_at 7.360626012
1415681_at 6.182229178
1415682_at 5.893955957
1415683_at 8.050101653
1415684_at 5.333176071
1415685_at 6.611418221
1415686_at 9.133717141
1415687_a_at 6.686628718
1415688_at 8.123992594
1415689_s_at 3.628100675

Total number of rows: 45101

Table truncated, full table size 1026 Kbytes.




Supplementary file Size Download File type/resource
GSM239950.CEL.gz 5.7 Mb (ftp)(http) CEL
Raw data provided as supplementary file
Processed data included within Sample table

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