Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Mapping RNA Polymerase II Occupancy in Embryonic Stem Cells

Data Global Transcriptional Repression by PRC2
Key Developmental Regulators Are Targets of PRC2
PRC2 and Highly Conserved Elements
Signaling Genes Are Among PRC2 Targets
Activation of PRC2 Target Genes During Differentiation
Supplementary Information

Chromatin Immunoprecipitations

We performed independent immunoprecipitations for each whole-genome analysis. Human WA09 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 that had been preincubated with 10 µg of the appropriate antibody. For cases where suppliers did not provide information regarding antibody concentration, 20 5l of the supplied solution was used per reaction. 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. Whole genome arrays were hybridized in batches of 35 to 60 arrays.

Slides were scanned using an Agilent DNA microarray scanner BA. PMT settings were set manually to normalize bulk signal in the Cy3 and Cy5 channel. For efficient batch processing of scans, we used Genepix (version 6.0) software. Scans were automatically aligned and then manually examined for abnormal features. Intensity data were then extracted in batch.

A more detailed experimental protocol can be found here.

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