Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation

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scan

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A mouse embryonic stem cell line was generated which stably expressed the ngn3 transcription factor under the control of the Tet-On inducible system using knock-ins in the ROSA26 and the HPRT loci. The undifferentiated mouse embryonic stem cells were then differentiated into Embryoid Bodies in suspension culture and were either treated with Doxycycline to induce NGN3 expression or left untreated as a contol. Cells were harvested at 12 hours, 24 hours and 48 hours.

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Transcription profiling by array of mouse embryonic stem cells expressing the transcription factor NGN3 for 12 hours, 24 hours and 48 hours

A mouse embryonic stem cell line was generated which stably expressed the ngn3 transcription factor under the control of the Tet-On inducible system using knock-ins in the ROSA26 and the HPRT loci. The undifferentiated mouse embryonic stem cells were then differentiated into Embryoid Bodies in suspension culture and were either treated with Doxycycline to induce NGN3 expression or left untreated as a contol. Cells were harvested at 12 hours, 24 hours and 48 hours.


csc.mrc.ac.uk:mimir/Protocol:AGVE_mESC_differentiation_01

csc.mrc.ac.uk:mimir/Protocol:hyb

Hybridisation of fragmented cRNA  Things to note: 1. There are two septa on the backside of the GeneChip.  Whilst filling or emptying the hybridisation chamber through one septum, a pipette tip must be inserted into the other as a vent. 2. The more you inset/remove tips into the septa, the looser they will become.  Eventually they will start to leak.  Method 1. Equilibrate array at room temperature for 30 minutes before use. 2. Heat frozen fragmented cRNA at 99oC for 5 minutes. 3. During incubation, pre-wet the array by filling with 200ul (100ul for test or mini arrays) of 1x hybridisation Buffer.  Incubate in rotisserie oven at 45oC, 60rpm for 10 minutes. 4. Cool fragmented cRNA at 45oC for 5 minutes. 5. To bring any particulate matter to the bottom of the tube, spin fragmented cRNA at maximum speed (?13,000g) for 5 minutes. 6. Remove buffer solution from array, ensuring that all solution is removed. 7. Add 200ul (100ul for test or mini array) of fragmented cRNA avoiding any debris at the bottom of the tube. 8. Hybridise in rotisserie oven at 45oC, 60rpm, for 16 hours (overnight). 9. Store remaining volume of fragmented cRNA at –20oC. 10. After hybridisation, proceed to “Preparation of GeneChip station”.  

csc.mrc.ac.uk:mimir/Protocol:hyb_cocktail

csc.mrc.ac.uk:mimir/Protocol:scan_3

A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation.

Transcription profiling by array of mouse embryonic stem cells expressing the transcription factor NGN3 for 12 hours, 24 hours and 48 hours

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