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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 01, 2010 |
| Title |
H3K36me3 ChIP-Seq-LSK cells R1 |
| Sample type |
SRA |
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| Source name |
Hematopoietic stem and progenitor cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Bone marrow isolated
antibody: H3K36me3
antibody manufacturer: Abcam
antibody catalog number: ab9050
antibody lot number: 761748
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| Growth protocol |
ES cells were grown in culture according usual labarotory practices. LSK cells were isolated from mouse bone marrow and FACS sorted for the presence of Lin-, Sca+, Kit+.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Low cell number ChIP-Seq library preparation: The library method described here consists of three main steps. Step 1: ChIP DNA (estimated at 10-50 picograms) is primed with a universal primer for four cycles (Primer 1, below) to create template DNA with common sequences incorporated at the ends. Primer 1 contains a common sequence for PCR, a restriction site for BciVI (underlined) and a random 9-mer at the 3’end. Priming is achieved using Sequenase V2.0 (US Biochemical 70775), a polymerase with strand displacement capability but no 3’→5’ exonuclease activity, as described7. First, we incubated 7 μl of ChIP DNA with 1 μl of Primer 1 (2 mM stock) and 2 μl of Sequenase buffer at 98 deg C briefly, and then annealed at 8 deg C for 5 min. We then added 5.5 μl of Sequenase Enzyme mix containing 1.5μl of 3nM dNTPs, 0.75μl 0.1M DTT, 1.5μl 500mg/ml BSA and 0.3μl 13U/μl Sequenase. The temperature was gradually increased to 370C and incubated for 8 min. The whole cycle was then repeated with the addition of 1.2 μl of diluted Sequenase (1:4) instead of the enzyme mix. After four cycles of priming, excess Primer 1 was cleaned up by exonuclease and alkaline phosphatase treatment as follows: 3 μl of ExoSAP-IT reagent (USB, Cat#: 78250) was added to 15 μl of step 1 reaction and incubated for 15 min at 37 deg C. The enzymes were then heat-inactivated at 85 deg C for 15 minutes. Step 2: Product from the priming reaction was PCR amplified using Primer 2 (below). The template DNA from step 1 was diluted by addition of 43 μl of water. Four parallel PCR reactions (50μl total volume each) were set up with 15 μl template DNA from Step 1, 1μl of Primer 2 (5 mM stock), 1.5μl of Phusion DNA Polymerase (NEB), 15μl of high GC buffer (NEB) and 1% DMSO. The amplification consisted of 15 cycles of denaturation (98 deg C for 30 sec), annealing (40 deg C for 30 sec and 50 deg C for 30 sec) and extension (72 deg C for 1 min). Step 3: PCR Product was column purified using the MinElute reaction clean up kit (Qiagen) and restricted overnight with BciVI (NEB) to yield 3’A overhangs at each end. Restricted fragments were ligated to Illumina adapters using the Quick Ligation Kit (NEB). This approach eliminates several standard library preparation steps, significantly reducing material loss. Ligation products were amplified for 18 cycles using standard Illumina primers and then size selected on 2 % agarose, retaining fragments between 275 and 600 bp. The resulting libraries were hybridized to flowcells, subjected to cluster amplification and sequenced on the Illumina Genome Analyzer using standard procedures. Primer 1: 5'-GACATGTATCCGGATGTNNNNNNNNN-3' Primer 2: 5'-GACATGTATCCGGATGT-3’
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Chromatin is immunoprecipitated with antibody against indicated epitope
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| Data processing |
Illumina reads were post-processed and aligned to the reference genome (UCSC, mm8) from the 10th base using MAQ. Reads mapped to more than one position in the genome were filtered out. Multiple reads mapping to the same position were only counted once to remove potential bias from PCR. Aligned reads were extended by 300 bases to roughly approximate the average size of sequenced fragments, and a 25 bp resolution density map was established by counting the number of fragments that overlap each position. Positions in the density map where less than 50% of the flanking 200 bp are alignable were masked as repetitive and discarded from further analysis.
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| Submission date |
Jun 01, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Jiang Zhu |
| E-mail(s) |
jzhu@mgh.harvard.edu
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| Organization name |
Massachusetts General Hospital, Harvard Medical School and Broad Institute
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| Lab |
Bradley Bernstein Lab
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| Street address |
185 Cambridge Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE22075 |
Genome-wide Chromatin Maps Derived from Limited Numbers of Hematopoietic Progenitors |
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| Relations |
| SRA |
SRX022212 |
| BioSample |
SAMN00015226 |
| Named Annotation |
GSM548826_mm8.LSK-RABr1.H3K36me3.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM548826_30DDNAAXX_6.dat.gz |
146.1 Mb |
(ftp)(http) |
DAT |
| GSM548826_mm8.LSK-RABr1.H3K36me3.wig.gz |
178.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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