Forum: Sample Prep / Library Generation
01-19-2021, 10:30 AM
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Replies: 289
Views: 154,980
Hi Luke,
thatīs correct, itīs not a mistake....
Hi Luke,
thatīs correct, itīs not a mistake. When starting from 50-150 pg cDNA you donīt need to use the standard amount of i5/i7. We generally dilute the adaptors 1:5 with water or low-EDTA TE...
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Forum: Sample Prep / Library Generation
07-29-2020, 01:22 AM
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Replies: 0
Views: 1,342
Superscript IV bacterial contamination?
Dear all,
we have recently performed single-cell RNA-seq using a variety of methods and enzymes (Smart-seq2, Smart-seq3, Takara, etc). We observed that when using Superscript IV the number of reads...
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Forum: Sample Prep / Library Generation
03-04-2020, 02:47 AM
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Replies: 289
Views: 154,980
Hi,
actually someone is still reading this...
Hi,
actually someone is still reading this thread :)
To answer your questions:
1- that's good!
2- 23-24 cycles is perfect, we never did more than that.
3- that might help as well. I would even...
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Forum: Sample Prep / Library Generation
07-27-2019, 02:55 AM
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Replies: 289
Views: 154,980
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Forum: Sample Prep / Library Generation
05-24-2019, 09:25 AM
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Replies: 289
Views: 154,980
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...
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Forum: Sample Prep / Library Generation
05-22-2019, 11:53 PM
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Replies: 289
Views: 154,980
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...
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Forum: Sample Prep / Library Generation
11-19-2018, 09:08 AM
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Replies: 3
Views: 3,075
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...
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Forum: Sample Prep / Library Generation
10-03-2018, 01:31 PM
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Replies: 289
Views: 154,980
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Forum: Sample Prep / Library Generation
09-20-2018, 07:28 AM
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Replies: 4
Views: 2,039
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Forum: Sample Prep / Library Generation
06-09-2018, 02:26 AM
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Replies: 289
Views: 154,980
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Forum: Sample Prep / Library Generation
06-08-2018, 10:36 AM
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Replies: 289
Views: 154,980
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...
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Forum: Sample Prep / Library Generation
06-08-2018, 10:12 AM
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Replies: 289
Views: 154,980
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...
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Forum: Sample Prep / Library Generation
06-07-2018, 05:09 AM
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Replies: 7
Views: 3,358
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Forum: Sample Prep / Library Generation
05-03-2018, 04:45 AM
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Replies: 289
Views: 154,980
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...
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Forum: Sample Prep / Library Generation
05-01-2018, 12:49 AM
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Replies: 5
Views: 2,572
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...
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Forum: Sample Prep / Library Generation
04-13-2018, 04:07 AM
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Replies: 5
Views: 3,536
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
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Forum: Sample Prep / Library Generation
02-08-2018, 10:32 AM
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Replies: 5
Views: 1,730
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Forum: Sample Prep / Library Generation
02-08-2018, 10:13 AM
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Replies: 3
Views: 2,009
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...
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Forum: Illumina/Solexa
12-17-2017, 03:45 AM
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Replies: 76
Views: 44,711
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Forum: Sample Prep / Library Generation
12-09-2017, 06:29 AM
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Replies: 289
Views: 154,980
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...
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Forum: Sample Prep / Library Generation
11-22-2017, 04:54 AM
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Replies: 289
Views: 154,980
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...
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Forum: Illumina/Solexa
11-14-2017, 02:10 AM
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Replies: 14
Views: 25,628
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...
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Forum: Sample Prep / Library Generation
09-15-2017, 01:10 AM
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Replies: 3
Views: 2,082
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Forum: Sample Prep / Library Generation
08-18-2017, 09:04 AM
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Replies: 3
Views: 2,383
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Forum: Illumina/Solexa
08-04-2017, 02:23 PM
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Replies: 17
Views: 5,485
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...
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