Forum: Bioinformatics
12-10-2016, 09:55 PM
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Replies: 15
Views: 10,854
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Forum: Bioinformatics
12-10-2016, 04:09 AM
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Replies: 15
Views: 10,854
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...
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Forum: Bioinformatics
12-03-2016, 01:24 AM
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Replies: 0
Views: 1,414
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,...
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Forum: Bioinformatics
11-23-2016, 09:27 PM
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Replies: 15
Views: 10,854
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Forum: Sample Prep / Library Generation
10-27-2016, 08:01 PM
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Replies: 4
Views: 2,612
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...
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Forum: Sample Prep / Library Generation
10-26-2016, 08:09 PM
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Replies: 4
Views: 2,612
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...
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Forum: Sample Prep / Library Generation
10-26-2016, 02:00 AM
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Replies: 4
Views: 2,612
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Forum: Sample Prep / Library Generation
04-19-2016, 03:55 AM
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Replies: 289
Views: 154,736
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Forum: Sample Prep / Library Generation
03-08-2016, 03:30 AM
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Replies: 289
Views: 154,736
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...
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Forum: Sample Prep / Library Generation
01-27-2016, 06:33 AM
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Replies: 289
Views: 154,736
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...
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Forum: Sample Prep / Library Generation
01-21-2016, 03:04 AM
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Replies: 289
Views: 154,736
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...
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Forum: RNA Sequencing
12-31-2015, 05:22 AM
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Replies: 7
Views: 7,225
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...
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Forum: RNA Sequencing
12-30-2015, 07:36 PM
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Replies: 7
Views: 7,225
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...
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Forum: Sample Prep / Library Generation
12-21-2015, 07:13 PM
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Replies: 289
Views: 154,736
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...
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Forum: Sample Prep / Library Generation
11-25-2015, 05:49 PM
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Replies: 289
Views: 154,736
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Forum: Sample Prep / Library Generation
11-19-2015, 01:35 AM
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Replies: 9
Views: 8,428
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Forum: Sample Prep / Library Generation
10-16-2015, 12:36 AM
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Replies: 4
Views: 2,062
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...
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Forum: Sample Prep / Library Generation
10-05-2015, 10:40 PM
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Replies: 4
Views: 2,062
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...
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Forum: Sample Prep / Library Generation
10-05-2015, 08:47 PM
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Replies: 289
Views: 154,736
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...
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Forum: Sample Prep / Library Generation
06-15-2015, 11:50 PM
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Replies: 33
Views: 28,469
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Forum: Sample Prep / Library Generation
06-14-2015, 10:08 PM
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Replies: 33
Views: 28,469
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Forum: Sample Prep / Library Generation
06-14-2015, 07:14 PM
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Replies: 33
Views: 28,469
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...
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Forum: Sample Prep / Library Generation
06-14-2015, 06:33 AM
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Replies: 33
Views: 28,469
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Forum: Sample Prep / Library Generation
06-09-2015, 04:31 AM
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Replies: 33
Views: 28,469
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...
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Forum: Sample Prep / Library Generation
05-27-2015, 01:19 AM
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Replies: 5
Views: 2,863
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...
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