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Forum: Sample Prep / Library Generation 07-27-2019, 01:55 AM
Replies: 278
Views: 124,341
Posted By Simone78
I personally prefer HPLC-purified oligos when...

I personally prefer HPLC-purified oligos when working with single cells but desalted would work as well.
Best,
Simone
Forum: Sample Prep / Library Generation 05-24-2019, 08:25 AM
Replies: 278
Views: 124,341
Posted By Simone78
From what I can see you seem to do everything...

From what I can see you seem to do everything correctly. You might get something out of the degraded cDNA (yes, it seems degraded to me) but you will also have a very strong 3ībias. The Tn5...
Forum: Sample Prep / Library Generation 05-22-2019, 10:53 PM
Replies: 278
Views: 124,341
Posted By Simone78
Thatīs really strange, it is generally the...

Thatīs really strange, it is generally the opposite: betaine and MgCl2 gives better yield, even when only just one of the two is included. Could you post the Bioanalyzer plots to get an idea of how...
Forum: Sample Prep / Library Generation 11-19-2018, 08:08 AM
Replies: 3
Views: 2,112
Posted By Simone78
Hi, Smart-seq2 works for bulk samples, however...

Hi,
Smart-seq2 works for bulk samples, however 10K cells might be too much. Among the possible drawbacks:
- lysis might be incomplete. To avoid this I would increase the % of Triton in the LB. You...
Forum: Sample Prep / Library Generation 10-03-2018, 12:31 PM
Replies: 278
Views: 124,341
Posted By Simone78
Hi, in principle you could use the protocol for...

Hi,
in principle you could use the protocol for single cell as it is, provided that the input RNA is not too high. I donīt know exactly, maybe up to 1 ng. If you start form several ng you should...
Forum: Sample Prep / Library Generation 09-20-2018, 06:28 AM
Replies: 4
Views: 1,548
Posted By Simone78
I am a big fan of the Tecan Freedom Evo 200. For...

I am a big fan of the Tecan Freedom Evo 200. For medium/high throughput project it's fantastic. Our configuration includes the 96 and 384 head adaptor. Having a 4 channel LiHa arm (4 independent...
Forum: Sample Prep / Library Generation 06-09-2018, 01:26 AM
Replies: 278
Views: 124,341
Posted By Simone78
One could try to increase the amount of Triton or...

One could try to increase the amount of Triton or use other (non-ionic) detergents such as Tween-20, Igepal CA-630, NP-40 or BSA (as in Svec et al. 2013, PMID: 24224157). Maybe some other RNAse...
Forum: Sample Prep / Library Generation 06-08-2018, 09:36 AM
Replies: 278
Views: 124,341
Posted By Simone78
the peak on the left, in my opinion, is...

the peak on the left, in my opinion, is primers/dimers. Unused primers, either because there is no RNA (NC) or degraded RNA will be amplified and accumulate in the reaction. If the RNA is little or...
Forum: Sample Prep / Library Generation 06-08-2018, 09:12 AM
Replies: 278
Views: 124,341
Posted By Simone78
I agree, the RNA controls should be much better...

I agree, the RNA controls should be much better and give tons of cDNA, especially the 100 pg and 1 ng. Lots of degraded stuff or leftover primers in general. S2 and S7 are decent, though. S3 and S4...
Forum: Sample Prep / Library Generation 06-07-2018, 04:09 AM
Replies: 7
Views: 2,609
Posted By Simone78
I would also say that it is over-tagmented. Which...

I would also say that it is over-tagmented. Which Tn5 are you using, Nextera or home-made? Maybe there is too much Tn5 for the number of cells you are using?
Forum: Sample Prep / Library Generation 05-03-2018, 03:45 AM
Replies: 278
Views: 124,341
Posted By Simone78
Hi, I would say the reaction failed completely...

Hi,
I would say the reaction failed completely and what you see still in the well is gDNA. I would first try SS2 on some good quality RNA (10 pg, 100 pg, 1 ng or higher), to eliminate issues with...
Forum: Sample Prep / Library Generation 04-30-2018, 11:49 PM
Replies: 5
Views: 1,961
Posted By Simone78
I also see another problem here. A 113-bp...

I also see another problem here. A 113-bp fragments is very close to the cutoff of the beads. I am not sure youīll be able to fish it out in an efficient way. Moreover, trying to avoid the...
Forum: Sample Prep / Library Generation 04-13-2018, 03:07 AM
Replies: 5
Views: 2,901
Posted By Simone78
we use SeraMag beads (carboxyl beads,...

we use SeraMag beads (carboxyl beads, hydrophylic). With a 150 EUR bottle (15 ml) one can make 750 ml of bead solution. We follow the protocol detailed in this paper: PMID:22267522
Forum: Sample Prep / Library Generation 02-08-2018, 09:32 AM
Replies: 5
Views: 1,462
Posted By Simone78
I donīt have access to the paper you refer to but...

I donīt have access to the paper you refer to but vaguely remember it.
I have some comments and suggestions:
- why do you sort in such a large volume of buffer. We generally sort in 2 ul (384w...
Forum: Sample Prep / Library Generation 02-08-2018, 09:13 AM
Replies: 3
Views: 1,514
Posted By Simone78
It depends which protocol you want to use. If...

It depends which protocol you want to use.
If you buy a Chromium or do Seq-well/Drop-seq/InDrop/Nadia system, etc etc most likely you donīt need any robot since the library prep will be done in 1...
Forum: Illumina/Solexa 12-17-2017, 02:45 AM
Replies: 76
Views: 40,493
Posted By Simone78
DEpending on which fragments you want to retain...

DEpending on which fragments you want to retain and which ones you would like to get rid of. To remove short fragments (dimers/primers) use a 0.8-1:1 ratio beads:DNA. If you want to pull down...
Forum: Sample Prep / Library Generation 12-09-2017, 05:29 AM
Replies: 278
Views: 124,341
Posted By Simone78
it is not recommended to freeze and thaw them...

it is not recommended to freeze and thaw them more than 4 times, according to Ambion. Upon arrival we generate hundreds of tubes with 15 ul of our final dilution (1:4M or 1:40M). 15 ul is the volume...
Forum: Sample Prep / Library Generation 11-22-2017, 03:54 AM
Replies: 278
Views: 124,341
Posted By Simone78
Hi, there are multiple options. It mostly...

Hi,
there are multiple options. It mostly depends on which method you want to use for depletion: RNAse H, DSN method post-amplification, bead capture. HEreīs a quick summary but let me know if you...
Forum: Illumina/Solexa 11-14-2017, 01:10 AM
Replies: 14
Views: 19,334
Posted By Simone78
I think other threads have probably additional...

I think other threads have probably additional information but I can summarize some things here.
You are correct, the gap needs to be filled and it can be done in 2 ways:
1- by a Polymerase with...
Forum: Sample Prep / Library Generation 09-15-2017, 12:10 AM
Replies: 3
Views: 1,749
Posted By Simone78
Please find it attached. Best, Simone

Please find it attached.
Best,
Simone
Forum: Sample Prep / Library Generation 08-18-2017, 08:04 AM
Replies: 3
Views: 1,940
Posted By Simone78
Hi, thatīs something we are thinking as well. I...

Hi,
thatīs something we are thinking as well. I donīt see any reasons why it shouldnīt work. I mean, the 50 ul reactions recommended in these Illumina kits are ridiculously expensive! Thatīs why...
Forum: Illumina/Solexa 08-04-2017, 01:23 PM
Replies: 17
Views: 4,518
Posted By Simone78
hi Arthur 45, sorry for the misunderstanding....

hi Arthur 45,
sorry for the misunderstanding. You do need to clean up the cDNA after the first PCR (pre-amplification for the Smart-seq2 protocol). I am not really familiar with the latest version...
Forum: Sample Prep / Library Generation 07-21-2017, 08:14 AM
Replies: 6
Views: 1,889
Posted By Simone78
Hi, I donīt think it really matters if you do...

Hi,
I donīt think it really matters if you do 10 or 12 cycles. The largest bias, where you "lost" your transcripts (due to many reasons that would take too long to explain here) is anyway introduced...
Forum: Sample Prep / Library Generation 07-21-2017, 01:46 AM
Replies: 6
Views: 1,889
Posted By Simone78
Hi, yes, probably 1 ng input is too much for...

Hi,
yes, probably 1 ng input is too much for the Tn5 you add, especially when using the Nextera XT kit. This leads to under-tagmentation and libraries with long(er) average size. Moreover, I would...
Forum: Sample Prep / Library Generation 07-01-2017, 05:52 AM
Replies: 12
Views: 10,589
Posted By Simone78
There are many protocols for producing...

There are many protocols for producing "home-made" Ampure beads . The one I follow (Rohland & Reich, Genome Res 2012) uses NaN3 (sodium azide) as preservative at a final conc of 0.05%. Ampure beads...
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