BioMicroCenter:ChIP

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The BioMicro Center now supports preparation of Chromatin IP samples. Basics about Chromatin IP can be found at [http://en.wikipedia.org/wiki/Chromatin_immunoprecipitation this link.]
The BioMicro Center now supports preparation of Chromatin IP samples. Basics about Chromatin IP can be found at [http://en.wikipedia.org/wiki/Chromatin_immunoprecipitation this link.]
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== Diagenode IP-STAR ==
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== Automated ChIP ==
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The BioMicro Center support for ChIP experiments begins with automation of the hosts a Diagenode IP-Star automated ChIP system.
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[[Image:BMC_IPstar.jpg|thumb|right|200px|Diagenode IP-Star]]Chromatin IP is one of the more complicated methods available through the BioMicro Center. In order to make this method reproducible enough to be run in a core facility, the BioMicro Center has automated the process through the use of the Diagenode IP-Star. The IP-star begins with sonicated chromatin and antibody and proceeds through the blocking, hyrbidization, washing and reverse crosslinking steps. RNaseA, ProteinaseK and cleanup steps are performed in the BioMicro Center off instrument and yields are assessed by Bioanalyzer. These ChIP samples may be picked up for qPCR or sent directly to SPRI for library construction and sequencing (options are listed on submission forms). In the event that the ChIP samples fail QC, the samples will not be submitted to SPRI-works but will be charged for the IP.  
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Sonicated chromatin will undergo IP on the IPstar. RNase A, Proteinase K, Phenol Chloroform extraction and purification will be performed off of the instrument and submitted for QC on the Bioanalyzer. These ChIP samples may be picked up for qPCR or sent directly to SPRI for library construction and sequencing (options are listed on submission forms). Users will be contacted if ChIP samples fail QC. These samples will not be submitted to SPRI-works but will be charged for the IP.  
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The Diagenode IP-star is a single purpose robot designed to handle IPs. The IP-Star has a throughput of 8 samples per day. The samples can vary on cell types, antibodies or buffer conditions, though the timing must remain constant. The system mixes the samples by repeatedly pipetting them. To separate the beads from the supernatant, the IP-Star works by placing a magnet next to the tips, retaining the beads in the tip while allowing the rest of the chromatin or washes to be removed. Once the sample is eluted from the beads, the beads are removed from the sample by keeping the beads in the tip again.  
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The IP-star requires 5 million cells per IP. It should work with many different cell lysis / crosslinking conditions but we have validated the protocol below:
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'''Chromatin''' – Sonicated chromatin must be submitted at 5 million cells per 200uL volume. Antibodies must be submitted at the same time(at least 3ug of antibody per IP). We have done months of testing and highly recommend using the following sonication conditions:
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  1. Resuspend fixed cells in 10mL of [[LB1|LB1]] <br>
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  2. Rock cells at 4 degrees for 10 minutes <br>
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  3. Pellet cells at 4 degrees for 5 minutes at 1350g <br>
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  4. Remove supernatant and resuspend cells in 10mL of [[LB2|LB2]] <br>
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  5. Rock cells at 4 degrees for 10 minutes <br>
 +
  6. Pellet cells at 4 degrees for 5 minutes at 1350g <br>
 +
  7. Remove supernatant and resuspend cells in a volume of [[LB3|LB3]] where there is 5 million cells/200uL <br>
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      '''**Example:''' Resuspend 25 million cells in 1mL of [[LB3|LB3]]'''**'''
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    <br>
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  8. Sonicate cells <br>
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  9. Pellet debris at 4 degrees for 10 minutes at 20,000g <br>
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  10. Submit lysate for ChIP
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<br>
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Sonication conditions vary broadly between different cell types and different sonicators so you will want to optimize your conditions to produce material in the 100-500nt rage. We strongly recommend that you not freeze your lysate before submitting it for ChIP therefore, it is critical that you coordinate with BioMicro Center staff members to be sure that space is available on the IP-Star when your cells are ready.
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[[Image:CHipPresentation2.jpg|thumb|right|Marson A, et al. Cell 2008]]
 
== ChIP-seq ==
== ChIP-seq ==
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[[Image:CHipPresentation2.jpg|thumb|right|Marson A, et al. Cell 2008]]
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Chromatin Immunoprecipitation Sequencing (ChIP-Seq) combines ChIP with DNA sequencing, allowing researchers to identify the binding sites of DNA-associated proteins. It can be used as a more cost effective and higher quality alternative to whole genome microarray hybridization. You can find more information about Illumina's ChIP-Seq protocols and kits [http://www.illumina.com/technology/chip_seq_assay.ilmn here.] ChIP-Seq samples are typically run as 36nt or 40nt single-end runs on the GAIIx or HiSeq, respectively.
Chromatin Immunoprecipitation Sequencing (ChIP-Seq) combines ChIP with DNA sequencing, allowing researchers to identify the binding sites of DNA-associated proteins. It can be used as a more cost effective and higher quality alternative to whole genome microarray hybridization. You can find more information about Illumina's ChIP-Seq protocols and kits [http://www.illumina.com/technology/chip_seq_assay.ilmn here.] ChIP-Seq samples are typically run as 36nt or 40nt single-end runs on the GAIIx or HiSeq, respectively.

Revision as of 07:42, 1 June 2012

Image:BioMicroCenter-header6.jpg

The BioMicro Center now supports preparation of Chromatin IP samples. Basics about Chromatin IP can be found at this link.

Automated ChIP

Image:BMC IPstar.jpg
Diagenode IP-Star
Chromatin IP is one of the more complicated methods available through the BioMicro Center. In order to make this method reproducible enough to be run in a core facility, the BioMicro Center has automated the process through the use of the Diagenode IP-Star. The IP-star begins with sonicated chromatin and antibody and proceeds through the blocking, hyrbidization, washing and reverse crosslinking steps. RNaseA, ProteinaseK and cleanup steps are performed in the BioMicro Center off instrument and yields are assessed by Bioanalyzer. These ChIP samples may be picked up for qPCR or sent directly to SPRI for library construction and sequencing (options are listed on submission forms). In the event that the ChIP samples fail QC, the samples will not be submitted to SPRI-works but will be charged for the IP.

The Diagenode IP-star is a single purpose robot designed to handle IPs. The IP-Star has a throughput of 8 samples per day. The samples can vary on cell types, antibodies or buffer conditions, though the timing must remain constant. The system mixes the samples by repeatedly pipetting them. To separate the beads from the supernatant, the IP-Star works by placing a magnet next to the tips, retaining the beads in the tip while allowing the rest of the chromatin or washes to be removed. Once the sample is eluted from the beads, the beads are removed from the sample by keeping the beads in the tip again.

The IP-star requires 5 million cells per IP. It should work with many different cell lysis / crosslinking conditions but we have validated the protocol below:

Chromatin – Sonicated chromatin must be submitted at 5 million cells per 200uL volume. Antibodies must be submitted at the same time(at least 3ug of antibody per IP). We have done months of testing and highly recommend using the following sonication conditions:

 1. Resuspend fixed cells in 10mL of LB1 
2. Rock cells at 4 degrees for 10 minutes
3. Pellet cells at 4 degrees for 5 minutes at 1350g
4. Remove supernatant and resuspend cells in 10mL of LB2
5. Rock cells at 4 degrees for 10 minutes
6. Pellet cells at 4 degrees for 5 minutes at 1350g
7. Remove supernatant and resuspend cells in a volume of LB3 where there is 5 million cells/200uL
**Example: Resuspend 25 million cells in 1mL of LB3**
8. Sonicate cells
9. Pellet debris at 4 degrees for 10 minutes at 20,000g
10. Submit lysate for ChIP


Sonication conditions vary broadly between different cell types and different sonicators so you will want to optimize your conditions to produce material in the 100-500nt rage. We strongly recommend that you not freeze your lysate before submitting it for ChIP therefore, it is critical that you coordinate with BioMicro Center staff members to be sure that space is available on the IP-Star when your cells are ready.

ChIP-seq

Marson A, et al. Cell 2008
Marson A, et al. Cell 2008

Chromatin Immunoprecipitation Sequencing (ChIP-Seq) combines ChIP with DNA sequencing, allowing researchers to identify the binding sites of DNA-associated proteins. It can be used as a more cost effective and higher quality alternative to whole genome microarray hybridization. You can find more information about Illumina's ChIP-Seq protocols and kits here. ChIP-Seq samples are typically run as 36nt or 40nt single-end runs on the GAIIx or HiSeq, respectively.



ChIP Seq Literature

  1. Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, Guenther MG, Johnston WK, Wernig M, Newman J, Calabrese JM, Dennis LM, Volkert TL, Gupta S, Love J, Hannett N, Sharp PA, Bartel DP, Jaenisch R, and Young RA. . pmid:18692474. PubMed HubMed [Paper1]
  2. Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, Zeng T, Euskirchen G, Bernier B, Varhol R, Delaney A, Thiessen N, Griffith OL, He A, Marra M, Snyder M, and Jones S. . pmid:17558387. PubMed HubMed [Paper2]
All Medline abstracts: PubMed HubMed
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