|
| Status |
Public on Dec 31, 2011 |
| Title |
K562 NF-E2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
NF-E2 ChIP DNA from hemin induced K562 cells at 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: pro-Erythroid cells
chip antibody: NF-E2
chip antibody vendor: Abcam Company
chip antibody cat. number: ab34682
|
| Treatment protocol |
K562 cells were treated by hemin into erythroid differentiation for 48h
|
| Growth protocol |
K562 cells were cultured in RPMI1640
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x10E6 K562 cells were cross-linked with 1% formaldehyde for 10 min, washed in cold PBS buffer, resuspended in lysis buffer, and sonicated to obtain chromatin fragments between 200 bp and 1000 bp. Sonicated chromatin was resuspended in IP buffer and incubated overnight at 4°C with magnetic beads conjugated antibodies ( Santa Cruz Biotechnologies). The IP was then washed with lysis buffer, LiCl buffer and TE buffer, eluted in elution buffer. The DNA was then recovered by reversing the crosslinks, and purified by QIAGEN Purification Kit. An un-enriched sample of DNA was treated in a similar manner to serve as input. The immunoprecipitated DNA was tested for enrichment of control loci by qPCR. To obtain sufficient DNA for hybridization, purified ChIP and input DNA was amplified with the WGA amplification kit
|
| Label |
Cy5
|
| Label protocol |
Amplified genomic DNA from purified ChIP and input DNA was fluorescently labelled using the NimbleGen Dual-Color DNA Labeling Kit as the Cy5-ChIP and Cy3-input labelled DNA samples
|
| |
|
| Channel 2 |
| Source name |
NF-E2 ChIP input DNA from hemin induced K562 cells at 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: pro-Erythroid cells
|
| Treatment protocol |
K562 cells were treated by hemin into erythroid differentiation for 48h
|
| Growth protocol |
K562 cells were cultured in RPMI1640
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x10E6 K562 cells were cross-linked with 1% formaldehyde for 10 min, washed in cold PBS buffer, resuspended in lysis buffer, and sonicated to obtain chromatin fragments between 200 bp and 1000 bp. Sonicated chromatin was resuspended in IP buffer and incubated overnight at 4°C with magnetic beads conjugated antibodies ( Santa Cruz Biotechnologies). The IP was then washed with lysis buffer, LiCl buffer and TE buffer, eluted in elution buffer. The DNA was then recovered by reversing the crosslinks, and purified by QIAGEN Purification Kit. An un-enriched sample of DNA was treated in a similar manner to serve as input. The immunoprecipitated DNA was tested for enrichment of control loci by qPCR. To obtain sufficient DNA for hybridization, purified ChIP and input DNA was amplified with the WGA amplification kit
|
| Label |
Cy3
|
| Label protocol |
Amplified genomic DNA from purified ChIP and input DNA was fluorescently labelled using the NimbleGen Dual-Color DNA Labeling Kit as the Cy5-ChIP and Cy3-input labelled DNA samples
|
| |
|
| |
| Hybridization protocol |
The Cy5-ChIP and Cy3-input labelled DNA samples were co-hybridized to NimbleGen HG18 RefSeq promoter arrays for 18 hours, post hybridization washes were carried out.
|
| Scan protocol |
microRNA promoter arrays were scanned using an Axon 4000B microarray scanner with GenePix 6.0.
|
| Data processing |
Raw data were extracted as pair files by NimbleScan software. We performed Median-centering, quantile normalization, and linear smoothing by Bioconductor packages Ringo, limma and MEDME. After normalization, a normalized log2-ratio data (*_ratio.gff file) was created for each sample. From the normalized log2-ratio data, a permutation-based peak-finding algorithm provided by NimbleScan v2.5 (Roche-NimbleGen) was applied to find peaks which represent significant positive enrichment. NimbleScan detects peaks by searching for 4 or more probes whose signals were above the specified cutoff values, ranging from 90% to 15%, using a 500bp sliding window. The cutoff values are a percentage of a hypothetical maximum, which is the mean + 6 [standard deviation]. The ratio data is then randomized 20 times to evaluate the probability of “false positives”. Each peak is then assigned a false discovery rate (FDR) score based on the randomization. After getting the *_peaks.gff files, the identified peaks with an FDR ≤ 0.2 were mapped to genomic features: transcripts.
|
| |
|
| Submission date |
Jul 20, 2011 |
| Last update date |
Dec 31, 2011 |
| Contact name |
Jia Yu |
| E-mail(s) |
j-yu@ibms.pumc.edu.cn
|
| Organization name |
Peking Union Medical College
|
| Department |
Biochemistry
|
| Lab |
Yu
|
| Street address |
Dong Dan San Tiao #5
|
| City |
Bei Jing |
| ZIP/Postal code |
100005 |
| Country |
China |
| |
|
| Platform ID |
GPL13949 |
| Series (2) |
| GSE30808 |
ChIP-chip from 48h hemin induced K562 cells with GATA-1, EKLF, and NF-E2 antibodies |
| GSE30809 |
Systematical identification of GATA-1, EKLF, and NF-E2 regulated miRNAs by ChIP-on chip and microRNA microarray in K562 cells into erythroid differentiation |
|
