F*****e
发帖数: 182 | 1 我们用小鼠组织做某个转录因子的ChIP，最终目标是希望能做ChIP-Seq。现在ChIP的流
程做完之后由于DNA浓度太低（0.1-0.2ng/ul by Nanodrop），不知道整个ChIP过程是
否成功，是否拿到有效的DNA片段。这个转录因子没有非常确定的target gene，所以也
无法设计引物做ChIP-PCR。请问各位，还有什么方法可以确定产物中含有富集的DNA片
段？多谢！ |
m*********n
发帖数: 215 | 2 what....I mean, you do not even know what DNA sequence you want to be "
riched", how do you know they are "riched" without sequencing? Are you just
wanna generally look at what this TF binds in the whole genome, or you are
testing whether it binds to a specific gene?
Ok, 先做library来获得更多的DNA.
Then... do you know the consensus binding sequence of this TF?
If not, and given that you do not have a positive control, you really need
to send the library for deep sequencing, and compare to control IgG-ChIPed
DNA or input DNA to find the real "peaks". If you do find enriched peaks,
then go back and do ChIP-qPCR. |
F*****e
发帖数: 182 | 3 多谢回复。
我们就是因为没有可靠的binding site作为positive constrol，所以无法验证ChIP是
否work。否则做个ChIP-PCR就知道是否得到了有效的DNA。
在整个genome里面做TF的binding site也是我们的目标，能找到感兴趣的基因当然最好。
如果直接做deep sequencing的话，时间和费用是个问题。 |
s******s
发帖数: 13035 | 4 首先，这个浓度做Nanodrop没啥意义
其次，你练啥positive的control都不知道，怎么定义有效？扔出去做library吧
【在 F*****e 的大作中提到】
: 我们用小鼠组织做某个转录因子的ChIP，最终目标是希望能做ChIP-Seq。现在ChIP的流
: 程做完之后由于DNA浓度太低（0.1-0.2ng/ul by Nanodrop），不知道整个ChIP过程是
: 否成功，是否拿到有效的DNA片段。这个转录因子没有非常确定的target gene，所以也
: 无法设计引物做ChIP-PCR。请问各位，还有什么方法可以确定产物中含有富集的DNA片
: 段？多谢！
|
m*********n
发帖数: 215 | 5 Then you definitely have to make a library for sequencing...... if you get
decent amount of reads and peaks, you can use the data to calculate the
consensus binding sequence for the TF. >
好。
【在 F*****e 的大作中提到】
: 多谢回复。
: 我们就是因为没有可靠的binding site作为positive constrol，所以无法验证ChIP是
: 否work。否则做个ChIP-PCR就知道是否得到了有效的DNA。
: 在整个genome里面做TF的binding site也是我们的目标，能找到感兴趣的基因当然最好。
: 如果直接做deep sequencing的话，时间和费用是个问题。
|
d****7
发帖数: 109 | 6 Before lib prep., to increase confidence，run 10% elutes on western blot to
check whether the protein is successfully pulled down. However, western blot
cannot guarantee the work of ChIP.
【在 F*****e 的大作中提到】
: 我们用小鼠组织做某个转录因子的ChIP，最终目标是希望能做ChIP-Seq。现在ChIP的流
: 程做完之后由于DNA浓度太低（0.1-0.2ng/ul by Nanodrop），不知道整个ChIP过程是
: 否成功，是否拿到有效的DNA片段。这个转录因子没有非常确定的target gene，所以也
: 无法设计引物做ChIP-PCR。请问各位，还有什么方法可以确定产物中含有富集的DNA片
: 段？多谢！
|
y*********1
发帖数: 46 | 7 If you can't find any known target of your transcription factor(TF) in the
literature, you can test if that TF binds its own promoter. In our
experience, a TF will bind to its own promoter, most of the time. |
F*****e
发帖数: 182 | 8 多谢各位！
看来需要直接做deep sequencing才能知道了，这种情况下需要把input也同样
sequencing么？这个应该就是打碎以后的genomic DNA吧。另外，sequencing的结果是
否要和input结果比较后才能知道有没有enriched DNA fragment？ |
m*********n
发帖数: 215 | 9 OF COURSE you need to sequence input DNA... did you read my previous
comments..
You really need to talk to the sequencing facility people first before you
make you next move. Also after preping your library, you have to do a
quality control analysis before moving to deep-sequencing.
【在 F*****e 的大作中提到】
: 多谢各位！
: 看来需要直接做deep sequencing才能知道了，这种情况下需要把input也同样
: sequencing么？这个应该就是打碎以后的genomic DNA吧。另外，sequencing的结果是
: 否要和input结果比较后才能知道有没有enriched DNA fragment？
|
F*****e
发帖数: 182 | 10 多谢momoshuihan。
我们是ChIP新手，希望大牛们指导。问题是：
1.input DNA
2.IgG-ChIPed DNA
3.Sample anitbody-ChIPed DNA
这三种样品是否都需要做sequencing？DNA是否得到enrichment是和1比较还是2比较？
另外能介绍一下怎样做qulity control analysis么？多谢！！
【在 m*********n 的大作中提到】
: OF COURSE you need to sequence input DNA... did you read my previous
: comments..
: You really need to talk to the sequencing facility people first before you
: make you next move. Also after preping your library, you have to do a
: quality control analysis before moving to deep-sequencing.
|
w****l
发帖数: 229 | 11 We also did RNA polymerase II pull down as a positive control. |
m*********n
发帖数: 215 | 12 You can just use input DNA and Antibody-ChIPed DNA for sequencing. For
quality control, your sequencing core facility will run the test for you, e.
g. Bio-analyzer chip etc.
One thing just hit me... at least in our hand, the DNA fragments needs to be
smaller for sequencing than for qPCR... I assume that you checked your
sonication efficiency and know the size of your smears before you did ChIP..
. Again, the best way is to talk to your sequencing facility and ask them
for technical details. This is rather important and will be more informative
than our advice here, because every facility might run samples differently.
Your facility core may even prep the library for you, if you did not have
any experience before. After the library is preped, they can run the
bioanalyzer first to check the size of your samples, the concentration as
well as the DNA integrity/purity etc etc...
PoII-ChIP is usually used as an indication that you can really do ChIP
experiments with these samples (to exclude the possibility of bad protocol,
bad protein protection etc.). But if you have obtained a resonable amount of
DNA (considering your starting cells/tissue), it should be fine.
【在 F*****e 的大作中提到】
: 多谢momoshuihan。
: 我们是ChIP新手，希望大牛们指导。问题是：
: 1.input DNA
: 2.IgG-ChIPed DNA
: 3.Sample anitbody-ChIPed DNA
: 这三种样品是否都需要做sequencing？DNA是否得到enrichment是和1比较还是2比较？
: 另外能介绍一下怎样做qulity control analysis么？多谢！！
|
w*******i
发帖数: 477 | 13 ChIP 完了，我们用Qubit 2.0 Fluorometer来测浓度。然后用10ng的DNA 做prep，然后
ChIP-Seq. 不能用nanodrop测，确实没有意义，测不准 |
