f******k
发帖数: 856 | 1 我这个问题很奇特，我用Invitrogen的dynabeads做ChIP-seq，最后拉下来的DNA量总是
比较高，做qPCR也没有enrichment，但是用同样的protocol，换回agarose beads，就
很少碰到这种问题。这不合理啊，dynabeads按理说背景要比agarose beads低很多才对
啊？！不知道大家有没有碰到这种情况？我试过好几种洗的方法和试剂，比如最常用的
就是低盐洗一次，高盐洗一次,LiCl洗一次，TE洗两次。也试过用RIPA洗过，也试过大
量延长洗的时间，但是最后elute下来DNA的量还是比较高。
另外，能不能请推荐一个比较好的做转录因子ChIP-seq的盒子或者详细的homemade
protocol？我做的这个转录因子，比较low abundant，可是我的材料是primary tissue
，又没法一次拿到很多的细胞（一次运气好的话可以有50-100million）。看有的文章
说现在10million就可以做转录因子的ChIP-seq了，可是我从来没成功过。现在真是快
被逼死啦。。。。快救救我 ：（ | m******5
发帖数: 1383 | 2 ChIP-Seq 就是agroase beads好用稳定
我也不知道为什么。也曾经有和你一样的问题，后来决定不折腾了，结果能用稳定就行
tissue
【在 f******k 的大作中提到】
: 我这个问题很奇特，我用Invitrogen的dynabeads做ChIP-seq，最后拉下来的DNA量总是
: 比较高，做qPCR也没有enrichment，但是用同样的protocol，换回agarose beads，就
: 很少碰到这种问题。这不合理啊，dynabeads按理说背景要比agarose beads低很多才对
: 啊？！不知道大家有没有碰到这种情况？我试过好几种洗的方法和试剂，比如最常用的
: 就是低盐洗一次，高盐洗一次,LiCl洗一次，TE洗两次。也试过用RIPA洗过，也试过大
: 量延长洗的时间，但是最后elute下来DNA的量还是比较高。
: 另外，能不能请推荐一个比较好的做转录因子ChIP-seq的盒子或者详细的homemade
: protocol？我做的这个转录因子，比较low abundant，可是我的材料是primary tissue
: ，又没法一次拿到很多的细胞（一次运气好的话可以有50-100million）。看有的文章
: 说现在10million就可以做转录因子的ChIP-seq了，可是我从来没成功过。现在真是快
| f******k
发帖数: 856 | 3 我也是折腾了好久，打算放弃磁珠了。那请问用agarose你是如何解决salmon sperm干
扰测序的问题呢？当然可能你不用salmon sperm DNA blocked agarose beads?
【在 m******5 的大作中提到】
: ChIP-Seq 就是agroase beads好用稳定
: 我也不知道为什么。也曾经有和你一样的问题，后来决定不折腾了，结果能用稳定就行
:
: tissue
| m******5
发帖数: 1383 | 4 买一套好点的kit就不用想这些事了
salmon sperm其实蛮好，反正也map不上
【在 f******k 的大作中提到】
: 我也是折腾了好久，打算放弃磁珠了。那请问用agarose你是如何解决salmon sperm干
: 扰测序的问题呢？当然可能你不用salmon sperm DNA blocked agarose beads?
| f******k
发帖数: 856 | 5 那能麻烦给推荐一个kit吗？
【在 m******5 的大作中提到】
: 买一套好点的kit就不用想这些事了
: salmon sperm其实蛮好，反正也map不上
| k*****n
发帖数: 323 | 6 try washing your Dynabeads with PBS 0.5% BSA 3 times, then incubate
antibodies in PBS-BSA for two hour. After IP, make sure resuspend beads
totally during each washing step. I never using sperm DNA. I would suggest
you using Pol II monoantibody 4H8 as positive control. Several steps are
critical for ChIP, 1, crosslinking, always using fresh FA, like 16% FA from
Thermo. for cells 15 min crosslinking with rotation would be enough, quench
with 0.125 M glycine for 1 min. 2 sonication, try Branson sonicator if you
are not sure about bioruptor or Covaris, which are quite easy overshearing.
for Branson, test different time, 30% output 7~10 x 10s with 40s off would
give you a good size distribution for both qPCR and Seq. 3, Washing, if you
get too much DNA, try using RIPA washing 4 time, however, in most case, the
DNA concentration is too low to get precise results. and you can always get
a false high concentration caused by the pheno extraction. I would suggest
low salt twice, high salt and LiCl once each, and TE once for most TF. | k*****n
发帖数: 323 | 7 For Seq, don't using any sperm DNA! it will saturate majority of your reads,
although it won't map to your genome!
for qPCR it might be fine.
【在 f******k 的大作中提到】
: 那能麻烦给推荐一个kit吗？
| d***s
发帖数: 1062 | 8 收藏了。
用过diagenode的kit还不错，就是manual写的不是很清楚。不知道有没有更新过，好在
客服不错。 | f******k
发帖数: 856 | 9 Thanks very much for your input.
I actually tried washing the beads using RIPA multiple times and it did
reduce the DNA yield compared to using regular washing steps. However, the
overall DNA yield was still quite high. I've no idea why.
from
quench
.
you
【在 k*****n 的大作中提到】
: try washing your Dynabeads with PBS 0.5% BSA 3 times, then incubate
: antibodies in PBS-BSA for two hour. After IP, make sure resuspend beads
: totally during each washing step. I never using sperm DNA. I would suggest
: you using Pol II monoantibody 4H8 as positive control. Several steps are
: critical for ChIP, 1, crosslinking, always using fresh FA, like 16% FA from
: Thermo. for cells 15 min crosslinking with rotation would be enough, quench
: with 0.125 M glycine for 1 min. 2 sonication, try Branson sonicator if you
: are not sure about bioruptor or Covaris, which are quite easy overshearing.
: for Branson, test different time, 30% output 7~10 x 10s with 40s off would
: give you a good size distribution for both qPCR and Seq. 3, Washing, if you
| f******k
发帖数: 856 | 10 我试了他们的iDeal ChIP-seq kit for Transcription Factor。 也是最后DNA yield
高，他们也是用的magnetic beads....
【在 d***s 的大作中提到】
: 收藏了。
: 用过diagenode的kit还不错，就是manual写的不是很清楚。不知道有没有更新过，好在
: 客服不错。
| k*****n
发帖数: 323 | 11 In general, magnetic beads works better than agroase beads for ChIP, I have
not encounter your situation before. Don't put sperm DNA, Check you DNA size
, get the concentration through qubit rather than nanodrop
【在 f******k 的大作中提到】
: Thanks very much for your input.
: I actually tried washing the beads using RIPA multiple times and it did
: reduce the DNA yield compared to using regular washing steps. However, the
: overall DNA yield was still quite high. I've no idea why.
:
: from
: quench
: .
: you
| k*****n
发帖数: 323 | 12 and using Pol II antibody as control. there is no TF antibodies can work
better than Pol II, if you can still get huge amount DNA, then is the
protocol or bead issue. if not, then your own antibody has issue. without
right control, it is hard for troubleshooting. best |
|
