s*********y
发帖数: 387 | 1 使用shRNA的全基因组库，做细胞功能的丧失方面的筛选，使用flow做为assay readout.
目前在看MISSION® LentiPlex™ Pooled Libraries，有时用过的朋友给些
建议吧，谢谢！
当然，如果有其他牌子的Pooled libraries的经验或者建议，也非常的欢迎！
谢谢 | s******y
发帖数: 28562 | 2 这个做下来得多少钱啊？
readout.
【在 s*********y 的大作中提到】
: 使用shRNA的全基因组库，做细胞功能的丧失方面的筛选，使用flow做为assay readout.
: 目前在看MISSION® LentiPlex™ Pooled Libraries，有时用过的朋友给些
: 建议吧，谢谢！
: 当然，如果有其他牌子的Pooled libraries的经验或者建议，也非常的欢迎！
: 谢谢
| g*********s
发帖数: 85 | 3 what's money? They don't care | r***e
发帖数: 2539 | 4 Cellecta有customerized library，以前想让他们把library做在我们自己的lentivect
or上，不过后来没谈成。
他们好像有free的DECIPHER Project，不大清楚怎么搞。
readout.
【在 s*********y 的大作中提到】
: 使用shRNA的全基因组库，做细胞功能的丧失方面的筛选，使用flow做为assay readout.
: 目前在看MISSION® LentiPlex™ Pooled Libraries，有时用过的朋友给些
: 建议吧，谢谢！
: 当然，如果有其他牌子的Pooled libraries的经验或者建议，也非常的欢迎！
: 谢谢
| B******o
发帖数: 496 | 5 这个library是based on pLKO hairpin shRNA。 这种shRNA比较高效。
OpenBiosystem还有另一种miR based shRNA pooled library。不过俺试过的～20
shRNA
clones knockdown效率都很差。但是这个library已经被好几个lab用过了(Gregary
Hannon, Michael Green等).你可以权衡一下,呵呵
Good luck
readout.
【在 s*********y 的大作中提到】
: 使用shRNA的全基因组库，做细胞功能的丧失方面的筛选，使用flow做为assay readout.
: 目前在看MISSION® LentiPlex™ Pooled Libraries，有时用过的朋友给些
: 建议吧，谢谢！
: 当然，如果有其他牌子的Pooled libraries的经验或者建议，也非常的欢迎！
: 谢谢
| j********r
发帖数: 156 | 6 那个TRC弄出的也称为一代shRNA (by Broad Institue). Hannon, Elledge作的是二代
，as 楼上mentioned, it is based on a natural miRNA structure. Both libraries
have been used extensively to generate lots of high-profile papers。
Generation II的library出的文章似乎要多一些，光 Elledge就好多篇 (such as
Synthetic lethality with RAS, cancer essential genes, tumor suppressor REST
and PTPN12, etc).
顺便问一下，shRNA导致的any phenotype是不是很难去rescue by introducing back
shRNA-resistant gene. 昨天还在看science那两篇cancer essential genes的文章，
似乎都没对任何hit试图rescue。那位大虾解释一下！ | d******u
发帖数: 178 | 7 shRNA library的话，好像就Sigma和Thermo/OpenBiosystems两家有。这两家不过是
Broad和CSH这两个source的distributor，主要就是pLKO和pGIPZ两种骨架的shRNA.两种
我都用过，因为每个基因一般会order3-5个hairpin，基本上里面至少有一个的
knockdown是不错的。 | s******y
发帖数: 28562 | 8 作这种实验大概需要多少钱啊？比方说一个library 需要多少钱来买啊？
【在 B******o 的大作中提到】
: 这个library是based on pLKO hairpin shRNA。 这种shRNA比较高效。
: OpenBiosystem还有另一种miR based shRNA pooled library。不过俺试过的～20
: shRNA
: clones knockdown效率都很差。但是这个library已经被好几个lab用过了(Gregary
: Hannon, Michael Green等).你可以权衡一下,呵呵
: Good luck
:
: readout.
| l******u
发帖数: 936 | 9 "as 楼上mentioned" 这个句子太搞了，哈哈。
libraries
REST
【在 j********r 的大作中提到】
: 那个TRC弄出的也称为一代shRNA (by Broad Institue). Hannon, Elledge作的是二代
: ，as 楼上mentioned, it is based on a natural miRNA structure. Both libraries
: have been used extensively to generate lots of high-profile papers。
: Generation II的library出的文章似乎要多一些，光 Elledge就好多篇 (such as
: Synthetic lethality with RAS, cancer essential genes, tumor suppressor REST
: and PTPN12, etc).
: 顺便问一下，shRNA导致的any phenotype是不是很难去rescue by introducing back
: shRNA-resistant gene. 昨天还在看science那两篇cancer essential genes的文章，
: 似乎都没对任何hit试图rescue。那位大虾解释一下！
| s*********y
发帖数: 387 | 10 sunnyday are (a),
sigma ask for 13k, saying good for 6 experiments.
【在 s******y 的大作中提到】
: 作这种实验大概需要多少钱啊？比方说一个library 需要多少钱来买啊？
| | | s*********y
发帖数: 387 | 11 biomacro, thanks for the infor.
I will not consider openbiosystem, as heard many complains about its
efficacy.
can you explain more about this
"这个library是based on pLKO hairpin shRNA。 这种shRNA比较高效"
I also worry about that for one cell, it may be infected by more than
one type of virus, making the downstream identification of specific ones
tricky?
do you think the flow based assay will be sufficient?
thanks.
【在 B******o 的大作中提到】
: 这个library是based on pLKO hairpin shRNA。 这种shRNA比较高效。
: OpenBiosystem还有另一种miR based shRNA pooled library。不过俺试过的～20
: shRNA
: clones knockdown效率都很差。但是这个library已经被好几个lab用过了(Gregary
: Hannon, Michael Green等).你可以权衡一下,呵呵
: Good luck
:
: readout.
| s******y
发帖数: 28562 | 12 谢谢！
是病毒based 的吧？
好贵。。。但是有些实验还是用这个比较直接。
【在 s*********y 的大作中提到】
: sunnyday are (a),
: sigma ask for 13k, saying good for 6 experiments.
| s*********y
发帖数: 387 | 13 thanks, try to follow your post.
"rescue by introducing back
I would say this is very difficult, unless you focus on several of
interests hits and redesign the shRNA targeting UTRs...
One another question, have you tried to work on Generation one shRNAs?
libraries
REST | d*p
发帖数: 534 | 14 I am using Broad library. There are 3 major problems for all the available
libraries.
1st, huge shRNA variation in pool, which means you are going to lose some
shRNAs due to low signal.
2nd, readout, both microarray and deep sequencing are available, however, if
you can not avoid PCR on the hairpin, you will always have difficulty in
the secondary structure.
3rd, even 5 shRNAs per gene, you will still find a large number of genes
without effective shRNA.
If you only need to find some interesting genes, it definitely works.
In terms of rescue, you also can change the last nt of the aa code.
【在 s*********y 的大作中提到】
: thanks, try to follow your post.
: "rescue by introducing back
: I would say this is very difficult, unless you focus on several of
: interests hits and redesign the shRNA targeting UTRs...
: One another question, have you tried to work on Generation one shRNAs?
: libraries
: REST
| s*********y
发帖数: 387 | 15 thanks.
I would say the points of 1 and 3 are really fine if we just want to
identify some targets, instead of a thorough screening.
Can you help explain more on
"2nd, readout, both microarray and deep sequencing are available,
however, if
you can not avoid PCR on the hairpin, you will always have difficulty in
the secondary structure."
you mean the PCR for the hairpin sequence will be difficult?
available
some
however, if
in
genes
【在 d*p 的大作中提到】
: I am using Broad library. There are 3 major problems for all the available
: libraries.
: 1st, huge shRNA variation in pool, which means you are going to lose some
: shRNAs due to low signal.
: 2nd, readout, both microarray and deep sequencing are available, however, if
: you can not avoid PCR on the hairpin, you will always have difficulty in
: the secondary structure.
: 3rd, even 5 shRNAs per gene, you will still find a large number of genes
: without effective shRNA.
: If you only need to find some interesting genes, it definitely works.
| B******o
发帖数: 496 | 16 pLKO shRNA是那种很传统的小RNA加个环，属于人工做的hairpin。但KD效果不错，俺买
来的或者自己做的，总有几个有很高的KD效率。总之成功率比较高，用着放心。
你如果担心一个细胞多个virus感染，可以把MOI控制好。Sigma给你的virus应该是已经
测好titer的，你计算一下尽量用低MOI，比如MOI=1. 这样的话细胞基本都只有一个
virus。
Flow是比较高效的方法，如果你确信有比较好的marker可以收集你要的细胞。收集好了
以后送deep seq就知道有哪些shRNA在里面了。
不过deep seq你需要好的data processing的人，noise很多，得想办法滤掉
【在 s*********y 的大作中提到】
: biomacro, thanks for the infor.
: I will not consider openbiosystem, as heard many complains about its
: efficacy.
: can you explain more about this
: "这个library是based on pLKO hairpin shRNA。 这种shRNA比较高效"
: I also worry about that for one cell, it may be infected by more than
: one type of virus, making the downstream identification of specific ones
: tricky?
: do you think the flow based assay will be sufficient?
: thanks.
| d*p
发帖数: 534 | 17 Yes, the hairpin structure interferes PCR and sequencing. So part of the
readout will be biased due to different PCR efficiency. Since you only want
to get some interesting genes, it is still feasible to your purpose.
【在 s*********y 的大作中提到】
: thanks.
: I would say the points of 1 and 3 are really fine if we just want to
: identify some targets, instead of a thorough screening.
: Can you help explain more on
: "2nd, readout, both microarray and deep sequencing are available,
: however, if
: you can not avoid PCR on the hairpin, you will always have difficulty in
: the secondary structure."
: you mean the PCR for the hairpin sequence will be difficult?
:
| n**********s
发帖数: 14 | |
|
