p15ink4b dna methylation: partial cell line: KG-1 antibody: H3K27me3
Growth protocol
RPMI-1640 medium containing 10% FBS and penicillin-streptomycin
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation assays were conducted as previously described (1Komashko VM, Acevedo LG, Squazzo SL, Iyengar SS, Rabinovich A, O'Geen H, Green R, Farnham PJ.Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells. Genome Res. 2008 18:521-32). AML cell lines were fixed in 0.8% formaldehyde for 6 min at 25oC. Following nuclei lysis, these samples were sonicated using a probe sonicator (Misonix, Inc; Farmingdale, NY) on ice for 15 cycles (20 sec per cycle) with a power level 3 with 1 min intervals of cooling. AML clinical samples were fixed in 1% formaldehyde for 10 min at 25oC and sonicated as above for eight 20 second cycles. Chromatin sheering was monitored by agarose gel electrophoresis and optimized to a size range of 200bp- 600bp. Chromatin (150-200ug) was immunoprecipitated with rabbit polyclonal antibodies H3me3K27 (Upstate # 07–449), H3K4me3 (Abcam #ab8580), and EZH2 (Active Motif# 39103). A 10% aliquot of each sample was removed as an input fraction. ChIP enriched DNA and input DNA was amplified using the Genomeplex whole genome amplification kit (WGA2, Sigma).
Label
Cy5
Label protocol
Two micrograms of amplified ChIP DNA or amplified input DNA was labeled with Cy3 (input) or Cy5 (IP)-dUTP (Perkin Elmer, Boston, MA) using the CGH labeling kit (Invitrogen, Carlsbad, CA).
RPMI-1640 medium containing 10% FBS and penicillin-streptomycin
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation assays were conducted as previously described (1Komashko VM, Acevedo LG, Squazzo SL, Iyengar SS, Rabinovich A, O'Geen H, Green R, Farnham PJ.Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells. Genome Res. 2008 18:521-32). AML cell lines were fixed in 0.8% formaldehyde for 6 min at 25oC. Following nuclei lysis, these samples were sonicated using a probe sonicator (Misonix, Inc; Farmingdale, NY) on ice for 15 cycles (20 sec per cycle) with a power level 3 with 1 min intervals of cooling. AML clinical samples were fixed in 1% formaldehyde for 10 min at 25oC and sonicated as above for eight 20 second cycles. Chromatin sheering was monitored by agarose gel electrophoresis and optimized to a size range of 200bp- 600bp. Chromatin (150-200ug) was immunoprecipitated with rabbit polyclonal antibodies H3me3K27 (Upstate # 07–449), H3K4me3 (Abcam #ab8580), and EZH2 (Active Motif# 39103). A 10% aliquot of each sample was removed as an input fraction. ChIP enriched DNA and input DNA was amplified using the Genomeplex whole genome amplification kit (WGA2, Sigma).
Label
Cy3
Label protocol
Two micrograms of amplified ChIP DNA or amplified input DNA was labeled with Cy3 (input) or Cy5 (IP)-dUTP (Perkin Elmer, Boston, MA) using the CGH labeling kit (Invitrogen, Carlsbad, CA).
Hybridization protocol
3 ug DNA IP and Input was co-hybridized according to Agilent protocol 65 degrees, 48 h.
Scan protocol
Arrays were scanned on an Agilent scanner per manufacturer's protocol
Description
Input
Data processing
The value is log2R normalized using Agilent feature extraction
