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.