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Sample GSM1085656 Query DataSets for GSM1085656
Status Public on Dec 15, 2017
Title 11823
Sample type RNA
 
Channel 1
Source name S3a-MEF PSENdKO+hPSEN1 Asp
Organism Mus musculus
Characteristics strain/background: C57B6/J black × 129Sv
cell type: mouse embryonic fibroblasts
genotype: PSEN1&2-/-
genetic modification: retroviral infection with human PSEN1 D257A/D385A mutant (puromycin resistant)
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from 80% confluent monolayer of cells using TriPure reagent and chloroform extraction. (1) Homogenization: Culture cells in 10cm² dishes, Use 1ml TriPure reagent/10cm² dish (when appending too little TriPure reagent, the chances are higher to contaminate the RNA with genomic DNA), Pass cell lysates several times through a pipet, To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to the addition of chloroform, Let stand at RT until completely resolved ~5min, Vortex samples. (2) Phase separation: Add 0.2ml chloroform per 1ml TriPure reagent, Shake vigorously for 15sec by hand, Incubate 2-3min at RT, Spin 15min at 12,000rpm at 4°C, Three layers are formed (top layer = aqueous phase containing RNA; white interphase layer = containing DNA and protein; bottom layer = red organic phase containing DNA and protein). (3) RNA precipitation: Mix aqueous phase (top layer) with 0.5ml isopropanol per 0.7-0.75ml TriPure reagent (new eppe), Incubate 10min at RT, Spin 10min at 12,000rpm at 4°C, The RNA precipitate forms a gel-like pellet (often invisible before centrifugation) on the side and bottom of the tube. (4) RNA wash: Remove supernatant, Wash RNA pellet with at least 1ml 75% EtOH per 1ml TriPure reagent used for the initial RNA isolation step, Vortex, Spin 5min at 7,500xg at 4°C. (5) Redissolve the RNA: Air-dry RNA pellet (5-10min) (Do not let RNA pellet dry out completely as this will greatly decrease its solubility), Redisolve in RNAse free water, Pass solution a few times through a pipet tip and incubate 10min at 55°C-60°C to avoid problems with partially dissolved RNA, Expected yield for fibroblasts: 5-7µg from 1*106 cells. (6) Store RNA samples at -80°C (1µg needed for complete exp)
Label Cy5
Label protocol RNA concentration and purity were determined spectrophotometrically using the NanoDrop ND-1000 (NanoDrop Technologies), and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 100ng of total RNA spiked with 10 viral polyA transcript controls (Agilent) was converted to double-stranded cDNA in a reverse transcription reaction. Subsequently, the sample was converted to antisense cRNA, amplified and labeled with Cyanine 5-CTP (Cy5) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent).
 
Channel 2
Source name S1a-MEF Wild-type
Organism Mus musculus
Characteristics strain/background: C57B6/J black × 129Sv
cell type: mouse embryonic fibroblasts
genotype: wild-type
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from 80% confluent monolayer of cells using TriPure reagent and chloroform extraction. (1) Homogenization: Culture cells in 10cm² dishes, Use 1ml TriPure reagent/10cm² dish (when appending too little TriPure reagent, the chances are higher to contaminate the RNA with genomic DNA), Pass cell lysates several times through a pipet, To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to the addition of chloroform, Let stand at RT until completely resolved ~5min, Vortex samples. (2) Phase separation: Add 0.2ml chloroform per 1ml TriPure reagent, Shake vigorously for 15sec by hand, Incubate 2-3min at RT, Spin 15min at 12,000rpm at 4°C, Three layers are formed (top layer = aqueous phase containing RNA; white interphase layer = containing DNA and protein; bottom layer = red organic phase containing DNA and protein). (3) RNA precipitation: Mix aqueous phase (top layer) with 0.5ml isopropanol per 0.7-0.75ml TriPure reagent (new eppe), Incubate 10min at RT, Spin 10min at 12,000rpm at 4°C, The RNA precipitate forms a gel-like pellet (often invisible before centrifugation) on the side and bottom of the tube. (4) RNA wash: Remove supernatant, Wash RNA pellet with at least 1ml 75% EtOH per 1ml TriPure reagent used for the initial RNA isolation step, Vortex, Spin 5min at 7,500xg at 4°C. (5) Redissolve the RNA: Air-dry RNA pellet (5-10min) (Do not let RNA pellet dry out completely as this will greatly decrease its solubility), Redisolve in RNAse free water, Pass solution a few times through a pipet tip and incubate 10min at 55°C-60°C to avoid problems with partially dissolved RNA, Expected yield for fibroblasts: 5-7µg from 1*106 cells. (6) Store RNA samples at -80°C (1µg needed for complete exp)
Label Cy3
Label protocol RNA concentration and purity were determined spectrophotometrically using the NanoDrop ND-1000 (NanoDrop Technologies), and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 100ng of total RNA spiked with 10 viral polyA transcript controls (Agilent) was converted to double-stranded cDNA in a reverse transcription reaction. Subsequently, the sample was converted to antisense cRNA, amplified and labeled with Cyanine 3-CTP (Cy3) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent).
 
 
Hybridization protocol A mixture of purified and labeled cRNA (Cy3 label: 825 ng; Cy5 label: 825 ng) was hybridised on Agilent's Mouse Whole Genome v2 oligo microarray (Cat # G2519F-026655, Agilent) followed by (manual) washing, according to the manufacturer's procedures.
Scan protocol To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner with SuresScan High-Resolution Technology. Probe signals were quantified using Agilent's Feature Extraction software (version 10.7.3.1).
Description MEF PSEN1&2-/- +hPSEN1 D257A/D385A vs. MEF Wild-type
Data processing We use the Agilent processed signal values (i.e., feature gProcessedSignal for the Cy3 signal and rProcessedSignal for the Cy5 signal) from Agilent Feature Extraction software v10.7.3.1 and compute log2-(Cy5/Cy3)-ratios. We remove Agilent control probes, as well as probes with signals below background on all arrays (absent spots). To decide whether a signal is significantly above background, we use the features gIsPosAndSignif and rIsPosAndSignif, also provided by the Agilent software. In case of multiple probes for the same Agilent ID, log2-(Cy5/Cy3)-ratios are averaged.
 
Submission date Feb 21, 2013
Last update date Dec 15, 2017
Contact name Rekin's Janky
E-mail(s) Nucleomics.Bioinformatics@vib.be
Organization name VIB
Department Nucleomics Core
Street address Herestraat 49 Box 816
City Leuven
ZIP/Postal code B-3000
Country Belgium
 
Platform ID GPL11202
Series (1)
GSE44454 Presenilins mediate endosomal recycling via ARF6 to maintain proper lysosomal clearance

Data table header descriptions
ID_REF
VALUE Log2 (Cy5/Cy3) ratio

Data table
ID_REF VALUE
A_51_P100034 0.668897700847941
A_51_P100174 0.619419840578401
A_51_P100289 0.306470096808363
A_51_P100298 -0.426516352395307
A_51_P100309 0.0518221569514969
A_51_P100327 -1.18661423114339
A_51_P100347 0.103391527935679
A_51_P100537 -0.756649758736563
A_51_P100573 -0.20769190016963
A_51_P100624 -0.501228178840722
A_51_P100625 -1.12922326695393
A_51_P100768 0.0886187474003706
A_51_P100776 0.354108946126698
A_51_P100787 0.0538718934023516
A_51_P100828 -0.289265142551817
A_51_P100852 -0.643674571056567
A_51_P100991 -0.152992715934363
A_51_P100997 0.580257770307279
A_51_P101006 -0.0519630174990168
A_51_P101075 -0.480800930989665

Total number of rows: 37513

Table truncated, full table size 1171 Kbytes.




Supplementary file Size Download File type/resource
GSM1085656_252665512435_04.txt.gz 15.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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