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Search: Posts Made By: kobeho24
Forum: Bioinformatics 12-10-2016, 08:55 PM
Replies: 15
Views: 10,098
Posted By kobeho24
Frankly speaking, due to the short read length of...

Frankly speaking, due to the short read length of NGS technology, there isn't such a kit or workflow. And it's not practical to do it on 3rd gen seq platform, since the throughput is still too low...
Forum: Bioinformatics 12-10-2016, 03:09 AM
Replies: 15
Views: 10,098
Posted By kobeho24
I disagree with the point that nomalizing the...

I disagree with the point that nomalizing the data with library size leads to the assumption that the initial abundance are the same. I suppose it's just a matter of library quantity instead of the...
Forum: Bioinformatics 12-03-2016, 12:24 AM
Replies: 0
Views: 1,288
Posted By kobeho24
Single-cell RNA-seq normalization with ERCC

Hi everyone,
Does anyone have any recommendation on the single-cell rna-seq data normalization methods? I would like to do normalization across a mix population of cells, in particular,...
Forum: Bioinformatics 11-23-2016, 08:27 PM
Replies: 15
Views: 10,098
Posted By kobeho24
It is known that TMM normalization factors do not...

It is known that TMM normalization factors do not take into account library sizes, I think it will be problematic if your library sizes are diversed.

Gary
Forum: Sample Prep / Library Generation 10-27-2016, 07:01 PM
Replies: 4
Views: 2,458
Posted By kobeho24
I have no idea about that either. But I already...

I have no idea about that either. But I already super diluted the reaction containing limited amount of SDS prior to beads purification~ I suppose it should be the buffer issue, somehow illumina just...
Forum: Sample Prep / Library Generation 10-26-2016, 07:09 PM
Replies: 4
Views: 2,458
Posted By kobeho24
Hi Phillip, Thanks for your reply. It may...

Hi Phillip,
Thanks for your reply. It may explain part of my confusion. But how come I cannot get a detectable mount of DNA with the condition of using SDS prior to beads purification? That doesn't...
Forum: Sample Prep / Library Generation 10-26-2016, 01:00 AM
Replies: 4
Views: 2,458
Posted By kobeho24
Questions about Nextera kit/tagmentation

Hi folks,
Just wonder if anyone has an experience of using the Nextera kit or home-made tagmentation assay based on the Genome Research paper....
Forum: Sample Prep / Library Generation 04-19-2016, 02:55 AM
Replies: 282
Views: 141,175
Posted By kobeho24
Check this out! It works pretty well on our hand....

Check this out! It works pretty well on our hand.
http://openwetware.org/wiki/SPRI_bead_mix
http://seqanswers.com/forums/showthread.php?t=49507&highlight=ampure
Forum: Sample Prep / Library Generation 03-08-2016, 02:30 AM
Replies: 282
Views: 141,175
Posted By kobeho24
Post Hi seqal, For you first question, I read some...

Hi seqal,
For you first question, I read some literature (Chenchik et al. 1998)about template switching, which is the main mechanism of SMART-seq2. It suggests that reverse transcriptase can search...
Forum: Sample Prep / Library Generation 01-27-2016, 05:33 AM
Replies: 282
Views: 141,175
Posted By kobeho24
Smile Hi Simone, Between these two, which one's...

Hi Simone,
Between these two, which one's better? It seems that SMARTscribe is similar to SSII and Maxima H- is similar to SSIV. Besides, as SMARTscribe is 100U/ul while Maxima H- is 200U/ul, do we...
Forum: Sample Prep / Library Generation 01-21-2016, 02:04 AM
Replies: 282
Views: 141,175
Posted By kobeho24
Unhappy SSII contamination

Hi all,
Recently I’ve done 3 runs of single-cell RNA-seq (Smart seq2) trial. I just followed the original protocol. A highly reproducible background curve can be observed even in negative controls...
Forum: RNA Sequencing 12-31-2015, 04:22 AM
Replies: 7
Views: 6,761
Posted By kobeho24
Smile Hi Misha, From my point of view, technical...

Hi Misha,
From my point of view, technical noise in single cell experiment such as PCR bias should always be taken into account. As far as I know, ERCC and UMI are the only two options we have to...
Forum: RNA Sequencing 12-30-2015, 06:36 PM
Replies: 7
Views: 6,761
Posted By kobeho24
Single-cell RNA-seq with ERCC RNA Spike-In

Hi all,
I recently came across some single-cell rna-seq experiment, in which people always recommand using ERCC spike-ins for normalization. And I just wonder how much spike-ins should be added per...
Forum: Sample Prep / Library Generation 12-21-2015, 06:13 PM
Replies: 282
Views: 141,175
Posted By kobeho24
Thumbs up Hi Simone, Sounds really great that you do have...

Hi Simone,
Sounds really great that you do have a protocol. What I wonder is RT condition for non-fragmented RNA. Just incubate the RNA with TGIRT at proper temperature for 15 min? Looking forward...
Forum: Sample Prep / Library Generation 11-25-2015, 04:49 PM
Replies: 282
Views: 141,175
Posted By kobeho24
Smile Hi Simone, I don't know if you noticed that the...

Hi Simone,
I don't know if you noticed that the newly published TGIRT paper in the journal RNA. They use the TGIRT to do the RT of human plasma RNA. And I realized that there is a fragmentation...
Forum: Sample Prep / Library Generation 11-19-2015, 12:35 AM
Replies: 9
Views: 8,161
Posted By kobeho24
excellent! thanks for sharing such impressive...

excellent! thanks for sharing such impressive stuff. BTW, any preference of kit shown above? neb, kapa, clontech which one has the best performance?

cheers,
gary
Forum: Sample Prep / Library Generation 10-15-2015, 11:36 PM
Replies: 4
Views: 1,936
Posted By kobeho24
Lightbulb Hi Simone, Can the 35-40% transcipt detection...

Hi Simone,
Can the 35-40% transcipt detection sensitivity of SMART-seq2 due to the low efficiency of template switching instead of the low processivity of retroviral RT? If I remember correctly, the...
Forum: Sample Prep / Library Generation 10-05-2015, 09:40 PM
Replies: 4
Views: 1,936
Posted By kobeho24
Hi Simone, If I remember correctly, the...

Hi Simone,
If I remember correctly, the pre-amplification in MALBAC is a quasilinear amplification to get a relatively higher amount of DNA with high fidelity. Once the abundance is relatively high...
Forum: Sample Prep / Library Generation 10-05-2015, 07:47 PM
Replies: 282
Views: 141,175
Posted By kobeho24
Smile Hi Simone, Glad to know that you have already...

Hi Simone,
Glad to know that you have already tried the TGIRT on your hand. I wonder if 0.2% SDS can strip the sticky enzyme off the DNA efficiently. And have you tried SDS along with high...
Forum: Sample Prep / Library Generation 06-15-2015, 10:50 PM
Replies: 33
Views: 26,642
Posted By kobeho24
Definitely, it is very hard to adjust the flow...

Definitely, it is very hard to adjust the flow rates as the paper described. And the flow rate is not a critical parameter to the protein production if it is slow enough for ensuring the high...
Forum: Sample Prep / Library Generation 06-14-2015, 09:08 PM
Replies: 33
Views: 26,642
Posted By kobeho24
BTW, can you PM your email address to me? So that...

BTW, can you PM your email address to me? So that we can have more discussion about it.:)
Forum: Sample Prep / Library Generation 06-14-2015, 06:14 PM
Replies: 33
Views: 26,642
Posted By kobeho24
I didn't use a specific equipment for protein...

I didn't use a specific equipment for protein purification. Bascially, I just followed every single step of the Genome Research paper. Moreover, I haven't tried on-column assembly yet, cuz I used to...
Forum: Sample Prep / Library Generation 06-14-2015, 05:33 AM
Replies: 33
Views: 26,642
Posted By kobeho24
Up till now, I've already had seveal attempts....

Up till now, I've already had seveal attempts. Nothing's wrong except for the activity. And now I am kinda puzzled. May I ask where did you get the pTXB1-Tn5 plasmid? Is it from Addgene?
Forum: Sample Prep / Library Generation 06-09-2015, 03:31 AM
Replies: 33
Views: 26,642
Posted By kobeho24
Smile Hi Simone, I tried to produce the Tnp once...

Hi Simone,
I tried to produce the Tnp once again with the C3013 lately. But the strain seemed to reach plateau phase after the A600=1.3. No matter there is IPTG induction or not, same thing...
Forum: Sample Prep / Library Generation 05-27-2015, 12:19 AM
Replies: 5
Views: 2,658
Posted By kobeho24
The TTP Labtech's Mosquito is kind of nanoliter...

The TTP Labtech's Mosquito is kind of nanoliter liquid handler, some researchers have already performed Nextera XT on their hand with this dispensing platform in a final volume of 4ul for each. And...
Showing results 1 to 25 of 32

 


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