Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells

Mapping Transcription Factor Occupancy in Human Embryonic Stem Cells

• Growth Conditions
• Immunohistochemical Analysis of Pluripotency Markers
• Teratoma Formation
• Human ES Cell Quality Control
• Immunohistochemical Analysis of Pluripotency Markers
• Teratoma Formation
• Embryoid Bodies (EB)
• Technology and Protocols
• Antibodies
• Chromatin Immunoprecipitations
• Microarray Design

• Download Data
• Data Analysis

Human embryonic stem cells were grown to a final count of 5x10^7 – 1x10^8 cells for each location analysis reaction. Cells were chemically crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 15 minutes at room temperature. Cells were rinsed twice with 1xPBS and harvested using a silicon scraper and flash frozen in liquid nitrogen. Cells were stored at –80°C prior to use.

Cells were resuspended, lysed in lysis buffers and sonicated to solubilize and shear crosslinked DNA. Sonication conditions vary depending on cells, culture conditions, crosslinking and equipment. We used a Misonix Sonicator 3000 and sonicated at power 7 for 10 x 30 second pulses (90 second pause between pulses). Samples were kept on ice at all times.

The resulting whole cell extract was incubated overnight at 4°C with 100 µl of Dynal Protein G magnetic beads blocked with PBS/BSA and that had been preincubated with 6-10 5g of the appropriate antibody. The immunoprecipitation was allowed to proceed overnight.

Beads were washed 5 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with occasional vortexing and crosslinking was reversed by overnight incubation at 65°C. Whole cell extract DNA (reserved from the sonication step) was also treated for crosslink reversal.

Immunoprecipitated DNA and whole cell extract DNA were then purified by treatment with RNAseA, proteinaseK and multiple phenol:chloroform:isoamyl alcohol extractions. Purified DNA was blunted and ligated to linker and amplified using a two-stage PCR protocol. Amplified DNA was labeled and purified using Bioprime random primer labeling kits (Invitrogen, immunoenriched DNA was labeled with Cy5 fluorophore, whole cell extract DNA was labeled with Cy3 fluorophore).

Labeled DNA was mixed (5 – 6 µg each of immunoenriched and whole cell extract DNA) and hybridized to arrays in Agilent hybridization chambers for 20 hours at 40°C. Arrays were then washed and scanned.

A more detailed experimental protocol can be found here.