Affymetrix GeneChip Murine Genome U74Bv2 [MG_U74Bv2]

Affymetrix GeneChip Murine Genome U74Cv2 [MG_U74Cv2]

Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency

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R1 ESC were cultured for 18h and 72h without leukemia inhibitory factor

Time

Transcription profiling of R1 embryonic stem cells cultured for 18h and 72h without leukemia inhibitory factor

R1 ESC were cultured for 18h and 72h without leukemia inhibitory factor


Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-10273

ESC were thawed and cultured for two passages over 96 hours on irradiated fibroblasts. To prepare cells for differentiation they were harvested and washed as described above, resuspended in ESC maintenance medium, and pre-plated on tissue culture plates for one hour at 37 C, 5% CO2 to deplete contaminating MEF. At the end of this pre-plating step the non-adherent ESC were discarded and the loosely adherent ESC were collected by gently washing the surface of the tissue culture plate. Cells were pelleted by centrifugation and viable cell numbers were determined. Contaminating MEF in the undifferentiated (day 0) ESC samples was estimated to be less than 0.2% based on cell size during counting.  These cells were used at the 0h time point

P-MEXP-10280

A portion of the pre-plated ESC were suspended at a density of 1-2 x 10^7 cells per 50 ml in liquid differentiation medium consisting of Iscove's modified Dulbecco's medium (IMDM), 15% FBS selected for its ability to support ESC differentiation, 2 mM glutamine, 150 uM MTG, and 40 ng/ml murine stem cell factor (STI) and plated into 4 x 100 mm Petri style culture dishes (Falcon). Cells were cultured overnight (18 hours) at 37 C, 5% CO2. The following morning embryoid bodies (EB), both in suspension and loosely attached, were harvested and allowed to settle to the bottom of a 50 ml conical tube for approximately 10 min. The supernatant, containing mainly single cells, was removed and the spontaneously pelleting EB fraction was collected by centrifugation at 1200 rpm for 7 min. EB were disrupted by incubation in T/E for 3 min at room temperature followed by passage through a 21-gauge needle to achieve single cell suspensions.  Following washing RNA was extracted.

P-MEXP-10281

Pre-plated ESC were plated at a density of 10^4 cells per 35 mm low-adherence Petri dish in IMDM-based ES differentiation methylcellulose consisting of 0.9% methylcellulose, 15% FBS, 2 mM glutamine, 150 uM MTG, and 40 ng/ml murine stem cell factor. Cultures were grown for 3 days and all EB in each dish were harvested by carefully flooding the dish with IMDM and collecting the methylcellulose/EB solution. EBs were washed twice in DMEM + 10% FBS to remove the residual methylcellulose and were then pooled and a single cell suspension generated.  RNA was extracted for analysis

P-MEXP-10283

P-MEXP-10313

Standard RMA normalization as implemented in the Affy package version 1.5.8 from Bioconductor.

P-MEXP-10347

Following two passages of growth on irradiated PEF post thaw, R1 ESC (p17) were transferred to relevant differentiation media (see specific treatment protocols) and grown at 37C/5% CO2.

Transcription profiling of R1 embryonic stem cells cultured for 18h and 72h without leukemia inhibitory factor

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