ChIP assays were performed on freshly dissected sciatic nerves, which were pooled from 6-10 P15 rat pups and minced in 1% formaldehyde in PBS as described (Jang et al., J. Neurochemistry, 2006; Jang and Svaren, JBC 2009). The in vivo FAIRE assay on rat myelinating sciatic nerve was performed at P14 using pooled nerves from 4 rat pups. Freshly dissected sciatic nerves were minced in phosphate-buffered saline (PBS) containing 1% formaldehyde for 5 minutes at room temperature (22oC). The nerves were washed with cold PBS, pooled, and frozen at -80oC. The nerves were thawed and homogenized in buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 8.0) containing protease inhibitor cocktail (Sigma, St Louis, MO, USA; 5µl of cocktail per ml of buffer). The cells were then lysed using incubation in three buffers, L1, L2 and L3 as previously described (Giresi and Lieb 2009). After cell lysis, cells were sonicated using the Bioruptor (Diagenode), and extracted with phenol:chloroform as previously described (Giresi and Lieb 2009). The protocol only differed in that the cells were sonicated in 2 ml of Lysis Buffer 3 on high with 30 seconds on followed by 30 seconds off, all on ice. 20% of the crosslinked chromatin was kept without the initial phenol extraction as an input control, and the samples were all reverse crosslinked at 65oC overnight.
Label
Cy5
Label protocol
ChIP DNA was amplified by whole genome amplification (Sigma), and then sent to Nimblegen for labeling with Cy3 or Cy5 per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
strain: Sprague-Dawley tissue: P15 sciatic nerve fraction: WGA-amplified input DNA
Extracted molecule
genomic DNA
Extraction protocol
ChIP assays were performed on freshly dissected sciatic nerves, which were pooled from 6-10 P15 rat pups and minced in 1% formaldehyde in PBS as described (Jang et al., J. Neurochemistry, 2006; Jang and Svaren, JBC 2009). The in vivo FAIRE assay on rat myelinating sciatic nerve was performed at P14 using pooled nerves from 4 rat pups. Freshly dissected sciatic nerves were minced in phosphate-buffered saline (PBS) containing 1% formaldehyde for 5 minutes at room temperature (22oC). The nerves were washed with cold PBS, pooled, and frozen at -80oC. The nerves were thawed and homogenized in buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 8.0) containing protease inhibitor cocktail (Sigma, St Louis, MO, USA; 5µl of cocktail per ml of buffer). The cells were then lysed using incubation in three buffers, L1, L2 and L3 as previously described (Giresi and Lieb 2009). After cell lysis, cells were sonicated using the Bioruptor (Diagenode), and extracted with phenol:chloroform as previously described (Giresi and Lieb 2009). The protocol only differed in that the cells were sonicated in 2 ml of Lysis Buffer 3 on high with 30 seconds on followed by 30 seconds off, all on ice. 20% of the crosslinked chromatin was kept without the initial phenol extraction as an input control, and the samples were all reverse crosslinked at 65oC overnight.
Label
Cy3
Label protocol
ChIP DNA was amplified by whole genome amplification (Sigma), and then sent to Nimblegen for labeling with Cy3 or Cy5 per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Hybridization protocol
Hybridization was performed by Nimblegen
Scan protocol
Arrays were scanned by Nimblegen
Description
custom array design 081105
Data processing
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
