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Sample GSM2309009 Query DataSets for GSM2309009
Status Public on Jan 01, 2017
Title REC_APB5_P8_C_rep1
Sample type RNA
Source name GFP-positive cells from P8 retina in APB5-treated homozygous Tie2-GFP transgenic mice
Organism Mus musculus
Characteristics age: postnatal day 8
tissue: retiina
cell type: endothelial
Treatment protocol Eyes from APB5-treated homozygous Tie2-GFP transgenic mice were enucleated, rinsed with PBS, and kept in staining solution (PBS containing 2mM EDTA, 0.01%, NaN3, 5% fetal calf serum, 50U/ml penicillin, and 0.05mg/ml streptomycin). The retinas were dissected under stereomicroscope, pooled in staining solution, and minced into small pieces. Collected retinal pieces were rinsed twice in PBS and digested in 10 ml of papain/DNase solution (papain (33U/ml; Sigma-Aldrich, St. Louis, MI) in Dulbecco’s PBS (Invitrogen) containing L-cysteine (0.4mg/ml; Nacalai tesque, Kyoto, Japan), EDTA (0.50mM; Sigma), and DNase I (125U/ml; Sigma)) for 60 min at 37°C with gentle agitation. Following digestion, cells were pelleted and treated two times with DNase/ovomucoid solution (a solution containing ovomucoid (2mg/ml; Sigma), DNase I (125 U/ml; Sigma), and BSA (1mg/ml; Wako, Osaka, Japan)) in Dulbecco’s PBS for 60 min at 37°C with gentle agitation. Digested retinal cells were filtered through nylon mesh, centrifuged at 300x g for 5 min to pellet cells, resuspended again with 10ml of DNase/ovomucoid solution incubated at 37°C for 60 min. Cells were rinsed twice in staining solution, pelleted, resuspended with staining solution containing 5µg/ml of propidium iodide, and filtered through nylon mesh. These cells were analyzed and sorted by FACS Aria (Becton Dickinson, San Jose, CA).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using RNeasy Micro-Kit (QIAGEN) and was amplified using MessageAmpII aRNA Kit (Ambion, Austin, TX) to obtain aRNA (>60μg).
Label biotin
Label protocol Biotin-labeled cRNA was synthesized from the obtained RNA sample using the BioArray RNA Transcript labeling Kit (Affymetrix, Santa Clara, CA).
Hybridization protocol Biotin-labeled cRNA was fragmented in a 40-μl reaction mixture containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30mM magnesium acetate, and incubated at 94°C for 35 min, and then hybridized onto the MGU74v2 series of arrays.
Scan protocol GeneChips were scanned with a GeneArray Scanner (Hewlett-Packard, Santa Clara, CA), controlled by MicroSuite 5.1 software (Affymetrix).
Description Gene expression data from GFP-positive cells from P8 retina in APB5-treated homozygous Tie2-GFP transgenic mice
Data processing The dChip software (version 1.3) was used to normalize the CEL files at probe level and compute model-based expression values using the PM only model.
Submission date Sep 12, 2016
Last update date Jan 01, 2017
Contact name Sentaro Kusuhara
Phone +81-78-382-6048
Organization name Kobe University Graduate School of Medicine
Department Department of Surgery
Lab Division of Ophthalmology
Street address 7-5-2 Kusunoki-cho, Chuo-ku
City Kobe
State/province Hyogo
ZIP/Postal code 650-0017
Country Japan
Platform ID GPL83
Series (1)
GSE86819 FACS-array profiling in retinal endothelial cells from living mouse retinas without pericyte coverage

Data table header descriptions
VALUE dChip normarization

Data table
132433_at 42.79
132434_at 14.86
132435_at 35.25
132436_f_at 61.36
132437_s_at 32.3
132443_at 176.71
132418_at 93.14
132405_s_at 105.24
132387_at 19.97
132388_f_at 160.87
132393_f_at 66.08
132397_at 64.03
132400_f_at 39.6
132403_at 139.47
132364_i_at 26.09
132365_r_at 65.86
132368_at 66.65
132370_at 52.95
132374_at 35.19
132381_at 52.4

Total number of rows: 11868

Table truncated, full table size 193 Kbytes.

Supplementary file Size Download File type/resource
GSM2309009_REC_APB5_1_MG_C_06042101.CEL.gz 2.5 Mb (ftp)(http) CEL
Processed data included within Sample table

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