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| Status |
Public on Aug 17, 2011 |
| Title |
Nelf-b_ChIPSeq |
| Sample type |
SRA |
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| Source name |
mouse embryonic fibroblast
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic fibroblast derived from Nelf-b f/- embryo
chip antibody: anti-Nelf-b (purified polyclonal)
strain: mixed background of 129/SvJ, C57BL/6, and FVB
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| Treatment protocol |
Asynchronously growing cells were used for ChIP
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| Growth protocol |
MEFs were maintained in Dulbecco's modification of Eagle's medium supplemented with 10% fetal bovine serum.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA was sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K, and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (60 g) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using the polyclonal anti-mouse Nelf-b antibody. After incubation at 4°C overnight, protein A-agarose beads were used to isolate the immune complexes. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR (qPCR) reactions were carried out in triplicate on specific genomic regions using SYBR Green Supermix (Bio-Rad). The resulting signals were normalized for primer efficiency by carrying out qPCR for each primer pair using Input DNA. ChIP and Input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3’ ends. Illumina adaptors were added and the library was size-selected (175-225 bp) on an agarose gel. The adaptor-ligated libraries were amplified for 18 cycles. The resulting amplified DNAs were purified, quantified, and tested by qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. Amplified DNAs (DNA libraries) were sent to Illumina Sequencing Services for sequencing on a Genome Analyzer II.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Chromatin IP against Nelf-b
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| Data processing |
Sequence aligns (35 bases) were extended in silico at their 3’ ends to a length of 110 bp, which is the average fragment length in the size-selected library. To identify the density of fragments (extended tags) along the genome, the genome is divided into 32-nt bins and the number of fragments in each bin is determined. This information is stored in a BAR (Binary Analysis Results) The MACS peak finding algorithm (Genome Biol, R137) was used to identify Nelf-b intervals, which represent the locations of fragment density peaks. A cut-off of p-value 1.00e-10 was applied and intervals presented in the Input sample were excluded. The identified Nelf-b intervals were compiled into BED file and further analyzed using Genpathway proprietary software that provides comprehensive information on genomic annotation, peak metrics, and sample comparisons for all peaks (intervals). Gene association of Nelf-b interval was analyzed by the “GeneMargin” method of Genpathway. The GeneMargin was set as a region 10,000 bp upstream and downstream of NCBI-annotated genes. Any interval within the GeneMargin of a gene is counted as being associated with that gene.
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| Submission date |
Sep 13, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Jianlong Sun |
| E-mail(s) |
jianlongs@gmail.com
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| Phone |
210-273-4879
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| Organization name |
Boston Children's Hospital
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| Department |
Hematology/Oncology
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| Lab |
Fernando Camargo
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| Street address |
1 Blackfan Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (2) |
| GSE24113 |
Regulation of Mammalian Cell Growth and Survival By an RNA Polymerase II-Pausing Factor (ChIP-seq) |
| GSE24180 |
Regulation of Mammalian Cell Growth and Survival By an RNA Polymerase II-Pausing Factor |
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| Relations |
| SRA |
SRX026745 |
| BioSample |
SAMN00113286 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM593398.bed.gz |
280.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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