Postnatal day 14-21 day mouse lateral ventricle walls were dissected and mechanically dissociated. Single cell suspensions were plated at low density (10,000~20,000 cells cm-2) on cell culture dishes coated with poly-L-ornithine (PO)/fibronectin (FN), allowing individual cells to form spatially distinct colonies. Serial passage (less than 2 passages, or 2 weeks) allowed us to enrich undifferentiated NSCs to high purity (>95% Nestin+/Sox2+). The NSCs were maintained in serum-free B27 medium in presence of 10 ng/ml FGF-2/bFGF (Peprotech) and 10 ng/ml EGF (Invitrogen). The monolayer cultures were serially passaged every 4-7 days. To avoid the potential non-specific effect induced by prolonged culturing, only NSCs with limited in vitro culturing (less than 3 weeks) were used in the study. J1 ESCs were maintained in standard culture conditions in presense of irradiated mouse embryonic fibroblasts.
Extracted molecule
genomic DNA
Extraction protocol
Each ChIP-chip assay was performed with about 20 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Dnmt3a (Santa Cruz, sc-20703), H3K4me3 (Millipore, 07-473), H3K27me3 (Millipore, 07-449), total histone H3 (Millipore, 07-690), Ezh2 (Active Motif, 39103) and Suz12 (A kind gift from Dr. Yi Zhang). MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight. Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C.
Label
Cy3
Label protocol
Whole cell extract and immunoprecipitated DNA were amplified using the Whole Genome Amplification Kit (Sigma), and 2 ug of amplified products was labeled with Cy3- and Cy5-dUTP (Perkin Elmer) using Bioprime DNA labeling system (Invitrogen).
Channel 2
Source name
KO NSC, immunoprecipitated DNA by H3K27me3 antibodies
Postnatal day 14-21 day mouse lateral ventricle walls were dissected and mechanically dissociated. Single cell suspensions were plated at low density (10,000~20,000 cells cm-2) on cell culture dishes coated with poly-L-ornithine (PO)/fibronectin (FN), allowing individual cells to form spatially distinct colonies. Serial passage (less than 2 passages, or 2 weeks) allowed us to enrich undifferentiated NSCs to high purity (>95% Nestin+/Sox2+). The NSCs were maintained in serum-free B27 medium in presence of 10 ng/ml FGF-2/bFGF (Peprotech) and 10 ng/ml EGF (Invitrogen). The monolayer cultures were serially passaged every 4-7 days. To avoid the potential non-specific effect induced by prolonged culturing, only NSCs with limited in vitro culturing (less than 3 weeks) were used in the study. J1 ESCs were maintained in standard culture conditions in presense of irradiated mouse embryonic fibroblasts.
Extracted molecule
genomic DNA
Extraction protocol
Each ChIP-chip assay was performed with about 20 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Dnmt3a (Santa Cruz, sc-20703), H3K4me3 (Millipore, 07-473), H3K27me3 (Millipore, 07-449), total histone H3 (Millipore, 07-690), Ezh2 (Active Motif, 39103) and Suz12 (A kind gift from Dr. Yi Zhang). MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight. Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C.
Label
Cy5
Label protocol
Whole cell extract and immunoprecipitated DNA were amplified using the Whole Genome Amplification Kit (Sigma), and 2 ug of amplified products was labeled with Cy3- and Cy5-dUTP (Perkin Elmer) using Bioprime DNA labeling system (Invitrogen).
Hybridization protocol
5 ug Cy3- or Cy5-labelled DNA were combined and hybridized to the Agilent 244K mouse extended promoter microarray (G4490A, 2-array set, genomic build: mm7) for 40 h at 65 ˚C (10 r.p.m.). Microarrays were washed according to Agilent's standard procedures.
Scan protocol
Microarry slides were scanned on at 5-micron resolution using an Agilent Technologies Scanner G2505B (US23502366).
Data processing
Final processed data file by ChIP Analytics 1.3: KO_NSC_H3K27me3_mm7_log2ratio.txt
Agilent Feature Extraction (FE) Software (v 9.5) was used for tiff image processing (VALUE providide in the Sample table below: log2 (Cy5_Signal/Cy3_Signal)). FE files (.txt) was subsequently analyzed using ChIP analytics 1.3 (Agilent). For IP/WCE experiments, we performed blank subtraction normalization, inter-array median normalization and intra-array median normalization by calculating a one-step Tukey biweight median. For IP/IP experiments, we performed an intra-array Lowess normalization. Replicate experiments were combined in ChIP analytics 1.3 (Agilent) to generate a weighted log2ratio for peak detection.
ChIP analytics data are provided as supplementary files on the Series record. The first three columns in the files represent genome coordinates (mm7) of all data probes with signals above the background threshold. The fourth column is the normalized log2 (Cy5_signal/Cy3_signal) for the corresponding probe calculated by ChIP Analytics. When replicate experiments available, the fourth column represents a weighted log2 ration from multiple experiments.
