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Forum: Sample Prep / Library Generation 04-08-2019, 07:29 AM
Replies: 18
Views: 3,021
Posted By arctan
Hi KB*, I agree with GenoMax. If you are going...

Hi KB*, I agree with GenoMax. If you are going to do NovaSeq, that is a huge amount of money (at least to me it is). I've only done MiSeq and NextSeq with short reads, so I don't have experience...
Forum: Sample Prep / Library Generation 04-04-2019, 08:47 AM
Replies: 18
Views: 3,021
Posted By arctan
Hi KB*, Adapter dimers can be a significant...

Hi KB*,

Adapter dimers can be a significant concern since they cluster and bridge-amplify efficiently and their small molecular weight means that a minor peak (in mass) can correspond to a fairly...
Forum: Sample Prep / Library Generation 04-03-2019, 10:06 PM
Replies: 18
Views: 3,021
Posted By arctan
Hi KB*, I agree with luc (but I am not that...

Hi KB*, I agree with luc (but I am not that experienced with this stuff), that the distribution of the library looks good.

I also cannot read the axes due to the low res images. I'm curious what...
Forum: Sample Prep / Library Generation 03-25-2019, 08:15 AM
Replies: 18
Views: 3,021
Posted By arctan
In some of my early library preps, I also saw and...

In some of my early library preps, I also saw and worried about a high molecular weight smear before. I thought it was bead carryover, but it wasn't.

I think it is daisy-chained library resulting...
Forum: Sample Prep / Library Generation 03-13-2019, 07:48 AM
Replies: 2
Views: 1,309
Posted By arctan
Thanks for replying! I am not sure what the...

Thanks for replying!

I am not sure what the peak is, but it seems reproducible because, I've seen it in all of the different RNA preps I've done where I also ran Bioanalyzer after rRNA-depletion....
Forum: Sample Prep / Library Generation 03-01-2019, 12:36 PM
Replies: 2
Views: 1,309
Posted By arctan
Prominent non-rRNA Bioanalyzer peak after rRNA depletion

Has anyone experienced seeing a prominent non-rRNA peak after rRNA-depletion? I am seeing this consistently in my samples.

See the attached image for an example, which shows Bioanalyzer RNA pico...
Forum: Sample Prep / Library Generation 06-07-2018, 11:30 AM
Replies: 10
Views: 2,649
Posted By arctan
Thank you both for the replies! As an...

Thank you both for the replies!

As an initial test, I acquired some Turbo DNase and treated an RIN >9 total RNA sample. I also had a positive control DNA-only sample to make sure the DNase is...
Forum: Sample Prep / Library Generation 06-04-2018, 10:42 AM
Replies: 10
Views: 2,649
Posted By arctan
Thanks for your reply. Since I am working with...

Thanks for your reply. Since I am working with bacterial RNA, splicing and introns aren't applicable. I agree with you though for eukaryotes, there can be many reads in introns in Ribo-Zero...
Forum: Illumina/Solexa 06-01-2018, 07:31 AM
Replies: 4
Views: 1,911
Posted By arctan
Well, I ended up getting the results of my run...

Well, I ended up getting the results of my run and the directionality (strand bias) is so bad and I reported about it here: http://seqanswers.com/forums/showpost.php?p=217710&postcount=5

Note to...
Forum: Sample Prep / Library Generation 06-01-2018, 07:25 AM
Replies: 10
Views: 2,649
Posted By arctan
Just wanted to follow up on this... Based on my...

Just wanted to follow up on this... Based on my recent experience, I think if you are doing RiboZero depletion (as opposed to Poly A selection, not an option for microbes), the library prep may be...
Forum: Illumina/Solexa 05-24-2018, 08:30 AM
Replies: 4
Views: 1,911
Posted By arctan
Thanks for the reply! I will keep that in mind...

Thanks for the reply! I will keep that in mind and a quick literature search does show significant antisense transcription in bacteria.

How would I distinguish real antisense transcription from...
Forum: Illumina/Solexa 05-23-2018, 06:36 PM
Replies: 4
Views: 1,911
Posted By arctan
Seeing reads in both directions from a directional library?

I made bacterial RNA-seq libraries with ScriptSeq v2 and did a small sequencing test (MiSeq Nano, Single Read, 50 bp).

ScriptSeq v2 prepares directional libraries, as per the manual: "The sequence...
Forum: Sample Prep / Library Generation 12-13-2017, 09:58 AM
Replies: 10
Views: 2,649
Posted By arctan
Thank you both for the helpful replies!

Thank you both for the helpful replies!
Forum: Sample Prep / Library Generation 12-11-2017, 08:20 AM
Replies: 10
Views: 2,649
Posted By arctan
genomic DNA contamination before RiboZero and ScriptSeq

This will be my first time making RNA-seq libraries. I have the RiboZero (for bacterial RNA) and ScriptSeq library prep kit.

I prepared bacterial total RNA and did on-column DNase treatment. ...
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