|
| Status |
Public on Dec 03, 2009 |
| Title |
ZNF263_ChIP-seq_A |
| Sample type |
SRA |
| |
|
| Source name |
K562 cells
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: ZNF263
cell type: myelogenous leukaemia
cell line: K562
passage: 10-15
|
| Growth protocol |
1. Take out the K562 vial (1 million cells in 1 ml) from liquid nitrogen and thaw it in 37 degree waterbath. Suspend the washed cells in 10 ml RPMI with 10% FBS and GIBCO Antibiotic-antimycotic (Cat. No. 15240-062 5ml per 500 ml of culture. 2. Centrifuge at 700 rpm for 5 min. 3. Suspend the cells in 10 ml RPMI with 10%FBS and transfer them into a small cell culture flask (not a spinner flask) to be incubated in CO2 incubator. 4. These cells get into log phase in 5 to 7 days. Start counting at day 3. When the cell density reaches 0.7 to 0.8(x 10?)/mlsplit the culture to about 0.4 million cells per ml with fresh RPMI with 10% FBS (this is the growth medium for K562). From this point on the cells should double every 24 hours, 5. From now on expect the cell density to double every 24 hours. (When the total cell number reaches 2X10>7, they can be stored as stock in liquid nitrogen at 1 million cells/ml (a total of 20 vials) in straight serum ( FBS) containing 10% DMSO) 6. Split the cells when the density reaches around 0.75 million/ml. 7. Grow the cells to required numbers.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Chromatin IP against ZNF263
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the human (March, 2006) genomes using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 400 bp to create summary windows.
Peaks were called with Sole-Search (http://chipseq.genomecenter.ucdavis.edu/cgi-bin/chipseq.cgi)
|
| |
|
| Submission date |
Dec 01, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Flora Vaccarino |
| Organization name |
Yale University
|
| Department |
Child Study Center
|
| Lab |
Vaccarino
|
| Street address |
230 South Frontage Road
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06520 |
| Country |
USA |
| |
|
| Platform ID |
GPL9052 |
| Series (1) |
| GSE19235 |
Genomic targets of the KRAB and scan domain-containing zinc finger protein 263 (ZNF263). |
|
| Relations |
| SRA |
SRX013323 |
| BioSample |
SAMN00005397 |
