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Forum: RNA Sequencing 07-11-2016, 07:55 AM
Replies: 1
Views: 1,014
Posted By sajoshi
Agilent small RNA chip ladder giving error

The ladder on the small RNA chip is giving error. We repeated a few times, but still the same. Why is that happening. The kit is not expired and ladder is not old either.

Please advise.
Forum: Illumina/Solexa 06-20-2016, 09:37 AM
Replies: 0
Views: 870
Posted By sajoshi
Has anyone done Zebrafish RNA-Seq?

I have attached RNA seq alignment results for Zebrafish and the % aligned is 65% (and I get 95% alignment with Human samples).

I wanted to know if you have done Zebrafish RNA-Seq and if you get...
Forum: Sample Prep / Library Generation 06-17-2016, 07:17 AM
Replies: 9
Views: 2,194
Posted By sajoshi
message

I just send you a message asking a question. Please let me know.
Forum: Sample Prep / Library Generation 06-17-2016, 06:43 AM
Replies: 0
Views: 805
Posted By sajoshi
small RNA library kit from Clontech

Has any one tried the Clontech kit for small RNA library prep. If so please provide feedback.

Thanks.
Forum: Illumina/Solexa 06-10-2016, 11:28 AM
Replies: 6
Views: 3,809
Posted By sajoshi
I quantify each library with Qubit, in...

I quantify each library with Qubit, in triplicates and take average. Then calculate how much you need for a 70 ng pool (for example). Change the pool concentration if needed to adjust final volume of...
Forum: Illumina/Solexa 06-10-2016, 11:14 AM
Replies: 4
Views: 2,100
Posted By sajoshi
Yes that is correct, ideally is 850 to 1000. I...

Yes that is correct, ideally is 850 to 1000. I agree with Genomax that clustering also depends on the sample type, library etc. Libraries do perform differently, so clustering will differ. Do not...
Forum: Sample Prep / Library Generation 06-10-2016, 07:38 AM
Replies: 9
Views: 2,194
Posted By sajoshi
small RNA library

See attached. Sample was purified when given to me and hence checked on smallRNA chip and BioA conc. was close to the conc. by Qubit microRNA assay. I used a human brain total RNA as control while...
Forum: Illumina/Solexa 06-10-2016, 07:24 AM
Replies: 2
Views: 985
Posted By sajoshi
HiSeq run results

Attached is a picture of the HiSeq 2500 run I recently did. The data looks really good. But the user says "I havenít mapped to the reference genome, but it does not look good. See the sequence...
Forum: Sample Prep / Library Generation 06-10-2016, 07:07 AM
Replies: 9
Views: 2,194
Posted By sajoshi
TruSeq smallRNA libraries

I am doing a TruSeq smallRNA library prep. When I was doing Ligate 3' adapter, and came to adding the STP step, the tube in the kit was empty (Illumina glitch). Illumina said to keep samples at -80...
Forum: Sample Prep / Library Generation 05-27-2016, 05:21 AM
Replies: 0
Views: 953
Posted By sajoshi
TruSeq smallRNA and chip-Seq libraries

1. I will be preparing smallRNA libraries using Illumna TruSeq kit. This is my first time and I was wondering if anyone has any feedback tips for the same. There is a gel excision part which I am not...
Forum: Sample Prep / Library Generation 05-12-2016, 01:35 PM
Replies: 2
Views: 959
Posted By sajoshi
How to get library size

Do my libraries look OK? Are they overloaded. How to I get the library size, I cant seem to adjust the upper and lower marker. Attached is the bioanalyzer profile. I called Agilent and they said...
Forum: Sample Prep / Library Generation 04-12-2016, 11:12 AM
Replies: 1
Views: 914
Posted By sajoshi
Bioanalyzer smallRNA results

Please see attached bioanalyzer profile for a smallRNA chip. I ran the same samples twice, but the ladder is giving some problem. I prepared the gel two times separately thinking that the gel might...
Forum: Illumina/Solexa 04-04-2016, 12:48 PM
Replies: 7
Views: 2,255
Posted By sajoshi
It was loaded at 15 pM and the clustering was...

It was loaded at 15 pM and the clustering was appropriate. Not over clustered at all. What conc. do you use?
Forum: Illumina/Solexa 04-04-2016, 10:51 AM
Replies: 3
Views: 1,981
Posted By sajoshi
16S metagenomics on MiSeq

I will be doing MiSeq run for 16S metagenomics. it will be my first time. Recently some MiSeq v3 300 PE runs gave me trouble and hence I am worried. Are there any things I should do differently from...
Forum: Illumina/Solexa 04-04-2016, 10:45 AM
Replies: 7
Views: 2,255
Posted By sajoshi
Do you mean under load library pool by 10%?

Do you mean under load library pool by 10%?
Forum: Illumina/Solexa 04-04-2016, 07:51 AM
Replies: 7
Views: 2,255
Posted By sajoshi
I think the libraries were prepared using...

I think the libraries were prepared using Nextera. I did not make them, it was another user running on our machine. The run failed three time at the 300 cycle. I think he spiked 5%. He has had...
Forum: Illumina/Solexa 04-04-2016, 05:35 AM
Replies: 7
Views: 2,255
Posted By sajoshi
MiSeq v3 PE 300 run and Illumina's service

MiSeq v3 PE 300 run failed the 3rd time for me. First time Illumina claimed that the software needs downgrading which we did. Second time they said problem is system fluidics, I checked that and it...
Forum: Sample Prep / Library Generation 03-15-2016, 12:33 PM
Replies: 7
Views: 3,102
Posted By sajoshi
What type of RNA would work best. I have seen...

What type of RNA would work best. I have seen sometimes CSF RNA samples have degraded RNA. I work in a core and asking other labs to donate samples for trying out the Clontech kit.
Forum: Sample Prep / Library Generation 03-15-2016, 05:35 AM
Replies: 7
Views: 3,102
Posted By sajoshi
I will probably use the Illumina protocol because...

I will probably use the Illumina protocol because we have Illumina HiSeq.
Forum: Sample Prep / Library Generation 03-10-2016, 11:39 AM
Replies: 7
Views: 3,102
Posted By sajoshi
Clontech Smart Seq v4 Ultra Low RNA Input

Have you used this kit? if you have used it what kind of RNA samples did you use. I have doing some initial experimentation and wondering which RNA would be suitable: RNA from paraffin (FFPE...
Forum: Sample Prep / Library Generation 03-04-2016, 11:14 AM
Replies: 2
Views: 2,541
Posted By sajoshi
Clean the pins with a brush. Alternate with...

Clean the pins with a brush. Alternate with propanol and milliq water a few times and try again.
Forum: Sample Prep / Library Generation 03-04-2016, 11:04 AM
Replies: 1
Views: 1,148
Posted By sajoshi
How to interpret this HS DNA library profile

Please see attached PDF and give your feedback. Are these good quality libraries for RNA seq.

Thanks,
Forum: Sample Prep / Library Generation 02-16-2016, 08:25 AM
Replies: 2
Views: 1,509
Posted By sajoshi
Thank you for the clarification

Thank you for the clarification
Forum: Sample Prep / Library Generation 02-11-2016, 11:11 AM
Replies: 2
Views: 1,509
Posted By sajoshi
EBT solution

I don't know if this is the right forum to ask. But I wanted to know the recipe for EBT solution using Tween. It cannot be ordered on it own from Illumina and I would to like to make it myself for...
Forum: Illumina/Solexa 01-07-2016, 11:35 AM
Replies: 1
Views: 944
Posted By sajoshi
HiSeq 2500 computer

I am trying to turn on the computer and its says no signal detected. This is stupid question but I am really new to this machine. How to start the computer.
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