Seeking advice on ChIP-Seq, RNA-Seq and/or miRNA array# Biology - 生物学
h*m
1 楼
唉，前阵子忙写论文，没有来版上多看两眼，这下亏了，还有SPG的卡申请不？
谢谢。。。。
谢谢。。。。
f*e
2 楼
第一个H1B 2015年7月中过期
5月初拿到 延期3年的H1B，到2018年8月中过期。
5月中回中国，上次签证还未过期1年，故中信代签---能否 代签网上有讨论，因为我的
H1B是新表了。
由于琐事缠身，到5月25日才得空去中信交材料
但是网上状态一直没有更新（我公婆交材料1周后，B签证都issued的了）
直到6月3日 状态才变。最后拿到护照已经是第15天（中信的小妹说一般不超2周）了。
回了美国才知道，系统出状况了。
好玄。如果我再晚交几天，签证不能打印，就不能按时回美国了，改机票又要花一笔。
5月初拿到 延期3年的H1B，到2018年8月中过期。
5月中回中国，上次签证还未过期1年，故中信代签---能否 代签网上有讨论，因为我的
H1B是新表了。
由于琐事缠身，到5月25日才得空去中信交材料
但是网上状态一直没有更新（我公婆交材料1周后，B签证都issued的了）
直到6月3日 状态才变。最后拿到护照已经是第15天（中信的小妹说一般不超2周）了。
回了美国才知道，系统出状况了。
好玄。如果我再晚交几天，签证不能打印，就不能按时回美国了，改机票又要花一笔。
r*1
3 楼
好可爱~
O*P
4 楼
就像霓虹灯到月亮的距离。
n*k
5 楼
Hey, folks:
We are planning to perform some ChIP/RNA-Seq as well as miRNA array
analysis some time soon. I was wondering any of you could kind of share
some of your experience in general.
Thanks a lot.
Something like: which services do you use(in house, or company), software,
etc etc. Which protocols, Kits? Pros and Cons, critical steps and tricks...
how many reads do you have, is it enough to cover the genome or enough for
the purpose? replicates? what about mRNA v lncRNA v miRNA, do you use polyA
/T column purification, or use Ribominus, or just total RNA; adapter or not,
amplification or not...arrays v seq....
In a word, any first hand experience/information on any of the steps/kits
would be highly appreciated....
BTW, we will use primary neural stem cell culture, therefore the starting
material likely is not a limiting factor.
We are planning to perform some ChIP/RNA-Seq as well as miRNA array
analysis some time soon. I was wondering any of you could kind of share
some of your experience in general.
Thanks a lot.
Something like: which services do you use(in house, or company), software,
etc etc. Which protocols, Kits? Pros and Cons, critical steps and tricks...
how many reads do you have, is it enough to cover the genome or enough for
the purpose? replicates? what about mRNA v lncRNA v miRNA, do you use polyA
/T column purification, or use Ribominus, or just total RNA; adapter or not,
amplification or not...arrays v seq....
In a word, any first hand experience/information on any of the steps/kits
would be highly appreciated....
BTW, we will use primary neural stem cell culture, therefore the starting
material likely is not a limiting factor.
x*g
6 楼
没啥亏的。你的论文比三万点重要得多。
deal总是有的，也许过几天会有更好的，到时候你就该庆幸没有跳以前的deal了，呵呵
deal总是有的，也许过几天会有更好的，到时候你就该庆幸没有跳以前的deal了，呵呵
c*8
7 楼
不错
【在 f******e 的大作中提到】
: 第一个H1B 2015年7月中过期
: 5月初拿到 延期3年的H1B，到2018年8月中过期。
: 5月中回中国，上次签证还未过期1年，故中信代签---能否 代签网上有讨论，因为我的
: H1B是新表了。
: 由于琐事缠身，到5月25日才得空去中信交材料
: 但是网上状态一直没有更新（我公婆交材料1周后，B签证都issued的了）
: 直到6月3日 状态才变。最后拿到护照已经是第15天（中信的小妹说一般不超2周）了。
: 回了美国才知道，系统出状况了。
: 好玄。如果我再晚交几天，签证不能打印，就不能按时回美国了，改机票又要花一笔。
r*1
8 楼
【在 r*********1 的大作中提到】
: 好可爱~
r*g
9 楼
哈哈，你让克肥唱中文歌试试，估计就倒过来了
e*t
10 楼
这个。。。
RNA seq，你送样品去测序不就行了，也不用你自己做。
重要的是随后的bioinfo，一般用bowtie做seq alignment分析。
RNA seq，你送样品去测序不就行了，也不用你自己做。
重要的是随后的bioinfo，一般用bowtie做seq alignment分析。
h*m
11 楼
呵呵。。。谢谢。
p*t
12 楼
满脑袋都是一瓶子不满半瓶子咣当。。。
O*P
13 楼
中国人都不唱中文歌了，人家为毛要唱中文歌啊？
n*k
14 楼
really?
no need to get rid of rRNA, do u care about ncRNA? Or the facility you
use will do all this for you? Could you please elaborate on your
experience? how about the pricing then?
thanks
【在 e*****t 的大作中提到】
: 这个。。。
: RNA seq，你送样品去测序不就行了，也不用你自己做。
: 重要的是随后的bioinfo，一般用bowtie做seq alignment分析。
no need to get rid of rRNA, do u care about ncRNA? Or the facility you
use will do all this for you? Could you please elaborate on your
experience? how about the pricing then?
thanks
【在 e*****t 的大作中提到】
: 这个。。。
: RNA seq，你送样品去测序不就行了，也不用你自己做。
: 重要的是随后的bioinfo，一般用bowtie做seq alignment分析。
b*u
15 楼
才看明白原来版标是个双鱼头小人啊~~
r*g
16 楼
所以说那英阴险呀。就姚贝娜的英语水平，这歌我女朋友唱都秒杀。
M*n
17 楼
if you outsource the job, you only need submit total RNA.
bioinfo can be done with a charge too. good thing about RNA-seq is that you
only need sample RNA and money to get a lot of data.
bioinfo can be done with a charge too. good thing about RNA-seq is that you
only need sample RNA and money to get a lot of data.
E*T
18 楼
金牛画得跟小猪似的
O*P
19 楼
不信
n*k
20 楼
what would be the price range?
I kind of like to do the bioinfo part myself for the seek of learning...
you
【在 M*****n 的大作中提到】
: if you outsource the job, you only need submit total RNA.
: bioinfo can be done with a charge too. good thing about RNA-seq is that you
: only need sample RNA and money to get a lot of data.
I kind of like to do the bioinfo part myself for the seek of learning...
you
【在 M*****n 的大作中提到】
: if you outsource the job, you only need submit total RNA.
: bioinfo can be done with a charge too. good thing about RNA-seq is that you
: only need sample RNA and money to get a lot of data.
o*1
21 楼
还好，不是最丑的。哈哈哈
m*p
22 楼
I spent about $600 for mRNA sequencing
w*e
23 楼
最后一个，还行
e*e
24 楼
where?
【在 m*p 的大作中提到】
: I spent about $600 for mRNA sequencing
【在 m*p 的大作中提到】
: I spent about $600 for mRNA sequencing
m*i
25 楼
果然哇。。。太可爱了！！
【在 r*********1 的大作中提到】
: 好可爱~
【在 r*********1 的大作中提到】
: 好可爱~
M*n
26 楼
i heard $1500 for one lane one run. bioinfo costs a couple hundred dollars.
if you want to save money, you may start with half-run.
【在 n********k 的大作中提到】
: what would be the price range?
: I kind of like to do the bioinfo part myself for the seek of learning...
:
: you
if you want to save money, you may start with half-run.
【在 n********k 的大作中提到】
: what would be the price range?
: I kind of like to do the bioinfo part myself for the seek of learning...
:
: you
n*k
27 楼
Could you please elaborate on it? thanks,
my research online and on the phone produces that most of the services ask
for 40-50k/sample if we just handle over the sample(total RNA or ChIP-DNA),
and they do the rest...or anywhere between 900-1500 per lane if they only
handle the sequencing part...
【在 m*p 的大作中提到】
: I spent about $600 for mRNA sequencing
my research online and on the phone produces that most of the services ask
for 40-50k/sample if we just handle over the sample(total RNA or ChIP-DNA),
and they do the rest...or anywhere between 900-1500 per lane if they only
handle the sequencing part...
【在 m*p 的大作中提到】
: I spent about $600 for mRNA sequencing
n*k
28 楼
Any friends out there have experience with any of them? I have reasonable
working knowledge but just don't have any experience and would appreciate
highly of your input, either here or through private communication...just
want to avoid major loss and get most out of our money as we are not as rich
as some such as peopblem or Morphin's lab...
..
polyA
not,
【在 n********k 的大作中提到】
: Hey, folks:
: We are planning to perform some ChIP/RNA-Seq as well as miRNA array
: analysis some time soon. I was wondering any of you could kind of share
: some of your experience in general.
: Thanks a lot.
: Something like: which services do you use(in house, or company), software,
: etc etc. Which protocols, Kits? Pros and Cons, critical steps and tricks...
: how many reads do you have, is it enough to cover the genome or enough for
: the purpose? replicates? what about mRNA v lncRNA v miRNA, do you use polyA
: /T column purification, or use Ribominus, or just total RNA; adapter or not,
working knowledge but just don't have any experience and would appreciate
highly of your input, either here or through private communication...just
want to avoid major loss and get most out of our money as we are not as rich
as some such as peopblem or Morphin's lab...
..
polyA
not,
【在 n********k 的大作中提到】
: Hey, folks:
: We are planning to perform some ChIP/RNA-Seq as well as miRNA array
: analysis some time soon. I was wondering any of you could kind of share
: some of your experience in general.
: Thanks a lot.
: Something like: which services do you use(in house, or company), software,
: etc etc. Which protocols, Kits? Pros and Cons, critical steps and tricks...
: how many reads do you have, is it enough to cover the genome or enough for
: the purpose? replicates? what about mRNA v lncRNA v miRNA, do you use polyA
: /T column purification, or use Ribominus, or just total RNA; adapter or not,
s*s
29 楼
找自己学校的facility吧。
这个$600就是批发折算的成本价，估计kit都要homemade才够。
$1k真的很便宜，不过没经验的做library可能也做不好。
),
【在 n********k 的大作中提到】
: Could you please elaborate on it? thanks,
: my research online and on the phone produces that most of the services ask
: for 40-50k/sample if we just handle over the sample(total RNA or ChIP-DNA),
: and they do the rest...or anywhere between 900-1500 per lane if they only
: handle the sequencing part...
这个$600就是批发折算的成本价，估计kit都要homemade才够。
$1k真的很便宜，不过没经验的做library可能也做不好。
),
【在 n********k 的大作中提到】
: Could you please elaborate on it? thanks,
: my research online and on the phone produces that most of the services ask
: for 40-50k/sample if we just handle over the sample(total RNA or ChIP-DNA),
: and they do the rest...or anywhere between 900-1500 per lane if they only
: handle the sequencing part...
p*7
30 楼
As far As I know, if the facility is very good at multiplexing, the cost can
get down significantly.
get down significantly.
n*k
31 楼
Thanks, Any tricks on constructing the library or any step along the line. I
have heard that contamination could be a huge problem, and trace of ethanol
is very bad etc etc...
Also, are there good software packages for data analysis and annotation,
such as strand specific analysis, long-non-coding RNA etc etc...
【在 s******s 的大作中提到】
: 找自己学校的facility吧。
: 这个$600就是批发折算的成本价，估计kit都要homemade才够。
: $1k真的很便宜，不过没经验的做library可能也做不好。
:
: ),
have heard that contamination could be a huge problem, and trace of ethanol
is very bad etc etc...
Also, are there good software packages for data analysis and annotation,
such as strand specific analysis, long-non-coding RNA etc etc...
【在 s******s 的大作中提到】
: 找自己学校的facility吧。
: 这个$600就是批发折算的成本价，估计kit都要homemade才够。
: $1k真的很便宜，不过没经验的做library可能也做不好。
:
: ),
n*k
32 楼
thanks...could you please elaborate a bit on that, and if possible, name a
few places which may have competitive pricing with service of high quality..
.Or anything u like to comment on the procedure, warning or tricks?
can
【在 p*****7 的大作中提到】
: As far As I know, if the facility is very good at multiplexing, the cost can
: get down significantly.
few places which may have competitive pricing with service of high quality..
.Or anything u like to comment on the procedure, warning or tricks?
can
【在 p*****7 的大作中提到】
: As far As I know, if the facility is very good at multiplexing, the cost can
: get down significantly.
c*r
33 楼
我做了四个RNA-seq样品，前两个是mRNA-seq，后两个是自己用invitrogen的Ribo-
Minus除去rRNA，期望能得到long ncRNA。样品是学校的Genomic core facility制备的
，一个大概$400多，测序和分析是在collaborator那里做的，目前没要钱，所以还不知
道他家的价钱。
但学校Genomic core facility的报价是：2x75 GA v5 kits：$2060/lane.
去年七月份在new mexico的一个institute做了一个咨询，价格大概是这样的：
Given:
- 2 libraries (you have already used the illumina SIPE kit to make paired
end library)
- Request for mRNA sequencing (PE)
- Sea urchin. genome size is ~850Mbases http://www.scienceonline.org/cgi/content/full/314/5801/941 so we know the transcriptome size will be much less. For example, if it is 10% of the genome (do you know?), that's ~85Mbases. If you wanted ~10x's coverage/sample, you would need ~850 Mbases/sample.
NOTE: These numbers & percentages you have to decide on yourself but it's
an example to help you understand the components of the design.
In this example, I would estimate 2 lanes of 2x36 per sample which should
yield ~800 or more Mbases per sample. However, I'll have to check to see
if we have other projects doing 2x36 with 4 lanes available to fill the 7
lane flow cell (see NOTE in pricing estimate below).
Question:
Do you know the size of the sea urchin Transcriptome and does one exist that
we can use for alignment? If the genome exists, we can use that.
Pricing estimate for example given:
$ 0 = 2 Short Insert Paired End Library preps (Customer provided)
$11600 = 4 lanes 2x36 mRNA sequencing. [NOTE: need to double check. 2x54
may be a viable option too.]
$ 2850 = Set-up ALPHEUS Variant and Expression Detection Pipeline w/ seq
urchin genome [use transcriptome too if available]
$ 3120= ALPHEUS Pipeline Run (one batch) against seq urchin genome/
transcriptome
$ 3514= 20% upcharge for ~4 week turn around
Minus除去rRNA，期望能得到long ncRNA。样品是学校的Genomic core facility制备的
，一个大概$400多，测序和分析是在collaborator那里做的，目前没要钱，所以还不知
道他家的价钱。
但学校Genomic core facility的报价是：2x75 GA v5 kits：$2060/lane.
去年七月份在new mexico的一个institute做了一个咨询，价格大概是这样的：
Given:
- 2 libraries (you have already used the illumina SIPE kit to make paired
end library)
- Request for mRNA sequencing (PE)
- Sea urchin. genome size is ~850Mbases http://www.scienceonline.org/cgi/content/full/314/5801/941 so we know the transcriptome size will be much less. For example, if it is 10% of the genome (do you know?), that's ~85Mbases. If you wanted ~10x's coverage/sample, you would need ~850 Mbases/sample.
NOTE: These numbers & percentages you have to decide on yourself but it's
an example to help you understand the components of the design.
In this example, I would estimate 2 lanes of 2x36 per sample which should
yield ~800 or more Mbases per sample. However, I'll have to check to see
if we have other projects doing 2x36 with 4 lanes available to fill the 7
lane flow cell (see NOTE in pricing estimate below).
Question:
Do you know the size of the sea urchin Transcriptome and does one exist that
we can use for alignment? If the genome exists, we can use that.
Pricing estimate for example given:
$ 0 = 2 Short Insert Paired End Library preps (Customer provided)
$11600 = 4 lanes 2x36 mRNA sequencing. [NOTE: need to double check. 2x54
may be a viable option too.]
$ 2850 = Set-up ALPHEUS Variant and Expression Detection Pipeline w/ seq
urchin genome [use transcriptome too if available]
$ 3120= ALPHEUS Pipeline Run (one batch) against seq urchin genome/
transcriptome
$ 3514= 20% upcharge for ~4 week turn around
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