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| Status |
Public on Nov 01, 2010 |
| Title |
negative2 |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LS174T
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Immunoprecipitated chromatin was sheared for a second time for 6 minutes using Covaris sonicator (6 x 16 mm AFA fiber Tube, duty cycle: 20%, intensity: 5, cycles/burst: 200, frequency sweeping) to obtain suitable shorter fragments (75-125 bp). To exclude a shearing bias as a possible source of binding site ultrastructure, partial digestion using DNaseI was used as an alternative to shorten the fragments for one control ChIP-seq library and one input library. For DNaseI treatment, chromatin was resuspended in 45 µl of freshly prepared reaction buffer (10mM MnCl2, 0.1mM CaCl2, 10mM Tris, pH 7.5) and digested with 0.5mU of DNaseI for 5 minutes at room temperature. The reaction was stopped by adding EDTA to a final concentration of 50mM. Chromatin was immediately extracted using phenol/chloroform procedure and precipitated. After fragmentation, fragments were blunt-ended and phosophorylated at the 5'-prime end using the End-it Kit (Epicentre) according to the manufacturer’s instructions. Ligation of double stranded adapters compatible with SOLiD sequencing was performed using Quick ligation kit (New England Biolabs) with 750 mM P1 ds and P2 ds adaptor (Applied Biosystems), 11.7 µl of 2x Quick ligation buffer, 1 µl Quick Ligase in total volume of 23.4 µl. Samples were purified using Ampure beads (Agencourt) and run on a native 6% polyacrylamide gel. Fragments ranging from 140 to 180 bp were excised; the piece of gel containing DNA fragments was shredded and dispersed into 400 µl of Platinum PCR Supermix with 750 mM of each P1 and P2 PCR primer, 2,5 U of Pfu (Stratagene) and 5 U Taq (Bioline). Prior to ligation-mediated PCR the sample was incubated at 72˚C for 20 minutes in PCR mix to let the DNA diffuse out of the gel and to perform nick translation on non ligated 3'-ends of DNA fragments. After 17 cycles of amplification the library was purified using Ampure beads and was quality checked on 2100 Bioanalyzer (Agilent) for the absence of possible adapter dimers and heterodimers.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 2.0 |
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| Description |
input DNA
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| Data processing |
Sequencing reads were quality trimmed by clipping at 3 consecutive nucleotides with quality score less than 10. Reads shorter than 18 nucleotides were discarded and the remaining reads were mapped against the human reference genome (hg18 assembly, NCBI build 36) using SHRiMP package (Rumble et al, 2009) with default settings, which allows mapping in SOLiD color space corresponding to dinucleotide encoding of the sequenced DNA. For analysis we used only uniquely placed reads, which were defined as reads having at least two additional mismatches in the second best hit compared to the best hit. The Cisgenome software package (Ji et al, 2008) was used for the identification of binding peaks from the ChIP-seq data. Two-sample analysis mode was used to compare samples with control input data. The parameters for peak discovery were set as follows: window size 100, step size 25, maximum gap 200, active single strand filtering with forward and reverse reads cutoff 4, minimum peak with 225 and minimum reads per window was set to obtain < 0.1 or 0.01 false positive rate (FDR).
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| Submission date |
Oct 08, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Michal Mokry |
| E-mail(s) |
m.mokry@umcutrecht.nl
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| Organization name |
Wilhelmina Children's Hospital, University Medical Center Utrecht
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| Street address |
Lundlaan 6
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| City |
Utrecht |
| ZIP/Postal code |
3584 EA |
| Country |
Netherlands |
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| Platform ID |
GPL9138 |
| Series (1) |
| GSE18481 |
Double fragmentation ChIP-seq provides single nucleotide resolution Tcf4 binding characteristics |
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| Relations |
| SRA |
SRX018546 |
| BioSample |
SAMN00010717 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM460127_Alignment_neg2.bed.gz |
40.7 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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