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Sample GSM4159087 Query DataSets for GSM4159087
Status Public on Jun 18, 2020
Title Pcp2-TRAP mouse cerebellum P21 replicate 2
Sample type RNA
 
Source name Pcp2-TRAP mouse cerebellum P21 replicate 2
Organism Mus musculus
Characteristics age: P21
Treatment protocol NA
Growth protocol All mice were healthy and were housed with a companion(s) in a specific-pathogen free animal facility in vented cages on a 12 hour light/dark cycle. Food and water were provided ad libitum.
Extracted molecule total RNA
Extraction protocol TRAP-RNA was isolated as previously described (Heiman et al., 2014; Kocabas et al., 2015; Zhu et al., 2016). Briefly, for Tg(Pcp2-L10a-Egfp) mice, pooled cerebella were immediately homogenized with a Teflon-glass homogenizer in ice-cold polysome extraction buffer (10 mM HEPES-KOH [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT (MilliporeSigma, D9779-1G), 100 µg/ml cycloheximide (MilliporeSigma, C7698-1G), Superasin and RNasin RNase inhibitors (ThermoFisher, AM2694, PR-N2515)and complete-EDTAfree protease inhibitor (MilliporeSigma, 11836170001)). Following clearing by centrifugation, supernatants were incubated at 4˚C with end-over-end rotation for 16-18 hours with biotinylated Streptavidin T1 Dynabeads (ThermoFisher, 65601) previously conjugated with GFP antibodies (Sloan Kettering Institute Antibody Core, HtzGFP-19C8 and HtzGFP-19F7). The beads were collected on a magnetic rack, washed, and resuspended in lysis buffer with β-mercaptoethanol (Agilent, 400753) to extract bound RNA from polysomes. RNA was purified using the Stratagene Absolutely RNA Nanoprep kit (Agilent, 400753). RNA quantity and quality was measured using an Agilent 2100 Bioanalyzer with the 6000 Pico Kit (Agilent, 5067-1513).
Label biotin
Label protocol 300 ng of total RNA was used to synthesize the first strand of cDNA using GeneChip WT Plus Reagent Kit from Affymetrix(P/N 902281). Single-stranded cDNA with T7 promoter sequence at the 5’ end was  synthesized in the reverse transcription reaction. The single-stranded cDNA was then converted into a double-stranded DNA (dsDNA) by DNA polymerase I in the presence of E. coli RNase H and DNA ligase. The dsDNA was then served as a template for amplification with T7 RNA polymerase to create antisense RNA. 15 μg of antisense RNA was used to generate sense-strand cDNA(ss-DNA) by reverse transcription using 2nd-Cycle Primers. The ss-cDNA contains dUTP at a fixed ratio relative to dTTP and 5.5 μg of ss-cDNA was fragmented by uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) at the unnatural dUTP residues. The fragmented ss-cDNA was labeled with biotin by terminal deoxynucleotidyl transferase (TdT).
 
Hybridization protocol 200 μl of hybridization solution containing 5.2 μg of the fragmented and labeled ss-DNA was hybridized to Affymetrix HTA 2.0 arrays (P/N 902662), (or  GeneChip™ Mouse Gene 1.0 ST Array  P/N 902464;  GeneChip™ Mouse Gene 2.0 ST Array P/N 902462) for 16 hours at 45°C. After hybridization, arrays were stained with streptavidin-phycoerythrin, followed by an antibody solution (anti-streptavidin) and a second streptavidin-phycoerythrin solution,( used The GeneChip™ Hybridization, Wash, and Stain Kit P/N 900720)  with all liquid handling performed by a GeneChip Fluidics Station 450. Arrays were then scanned with the Affymetrix GeneChip Scanner 3000 7G.
Scan protocol Mouse Gene 1.0 ST arrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
Data processing Normalised microarray expression values were acquired with Affymetrix Power Tools’s apt-probeset-summarize tool using rma-sketch normalisation,
MoGene-1_0-st-v1.r3.pgf
MoGene-1_0-st-v1.r3.mps
 
Submission date Nov 13, 2019
Last update date Jun 18, 2020
Contact name Tom Samuel Carroll
E-mail(s) thomas.carroll@rockefeller.edu
Organization name The Rockefeller University
Department Bioinformatics
Lab Bioinformatics
Street address 1230 York Avenue
City New York
State/province New York
ZIP/Postal code 10065
Country USA
 
Platform ID GPL6246
Series (2)
GSE140299 Expression data from Tg(Pcp2-L10a-Egfp) TRAP mice over postnatal mouse development. [Affymetrix 1]
GSE140307 Expression data from Tg(Pcp2-L10a-Egfp) TRAP mice over postnatal mouse development.

Data table header descriptions
ID_REF
VALUE RMA-sketch normalised expression values

Data table
ID_REF VALUE
10473562 4.77987
10385500 6.29567
10593927 12.08099
10457733 11.41521
10476889 9.03229
10438425 4.14156
10539444 7.04623
10424411 10.88875
10362379 9.482
10398100 6.53144
10517791 5.57737
10578690 5.56761
10418038 7.46967
10578922 9.35243
10442786 6.13857
10470665 10.51534
10364009 9.77079
10347106 10.07662
10583390 3.79749
10520304 10.08633

Total number of rows: 22092

Table truncated, full table size 369 Kbytes.




Supplementary file Size Download File type/resource
GSM4159087_P212_MoGene_1.0_ST_Xiaodong_091311.CEL.gz 4.6 Mb (ftp)(http) CEL
Processed data included within Sample table

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