[cmawhinney]

Hi all,

I am looking at the directional protocol now and see that it requires the v1.5 sRNA 3' Adaptor. Which adaptor is this? I'm assuming it is NOT the Small RNA 3' adaptor.

Thanks!

[som]

v1.5 sRNA 3'adapter is a new version 1.5 small RNA 3'adapter

The v1.5 small RNA 3' adapter is specifically modified to target microRNAs
and other small RNAs that have a 3' hydroxyl group resulting from enzymatic
cleavage by Dicer or other RNA processing enzymes (illumina).

for more information about this adapter

http://seqanswers.com/forums/showthread.php?t=1265

[protist]

Also do any of you guys know where illumina gets the 5x frag buffer and stop solution. Will buy you a beer if so :-)[/QUOTE]

The 5X frag buffer in the published protocol is not the same as the 10X Ambion product. 10X product is a buffered Zinc solution and fragmentation is at 70C - metal based fragmentation method. The 5x solution is based on the Affy buffer (TrisAcetate + KOAc + MgOAc) and fragmentation is carried out at the higher temp of 94C.

Both buffers work well - we have not switched from the Ambion buffer and have made many RNAseq libraries both non directional and directional without issue.

[cmawhinney]

v1.5 sRNA 3'adapter is a new version 1.5 small RNA 3'adapter

The v1.5 small RNA 3' adapter is specifically modified to target microRNAs
and other small RNAs that have a 3' hydroxyl group resulting from enzymatic
cleavage by Dicer or other RNA processing enzymes (illumina).

for more information about this adapter
http://seqanswers.com/forums/showthread.php?t=1265

This is exactly what I was looking for! Thank you so much!

[zorph]

i just finished doing this protocol. I did directional RNA-seq prep protocol with a DSN normalization to get rid of the rRNA.
I did get a 150bp product, but illumina said it was ok.

The protocols worked well according to the bioanalyzer, but just to double check, I cloned 32 fragments into a vector prior to placing it onto the sequencing machine to see if my product and to make sure everything was ligated properly--which it was. I had 50% transcripts and 50% rRNA which, according to Illumina's recommendation, was good enough to go onto the sequencer.

Also, if someone finds fragmentation buffer, please let me know!!!!

[som]

The 5X frag buffer in the published protocol is not the same as the 10X Ambion product. 10X product is a buffered Zinc solution and fragmentation is at 70C - metal based fragmentation method. The 5x solution is based on the Affy buffer (TrisAcetate + KOAc + MgOAc) and fragmentation is carried out at the higher temp of 94C.

Both buffers work well - we have not switched from the Ambion buffer and have made many RNAseq libraries both non directional and directional without issue.

I wonder how long the fragmented size you get from Ambion buffer.
I plan to perform 75-bp read, so the fragmented size i need is around 150-200b.
I have tried to optimize the fragmentation using Ambion buffer, but could get only around 100b.

Could you please share your results also the optimized condition.

Thank you

[Nik]

This is exactly what I was looking for! Thank you so much!

v1.5 sRNA 3'adapter is a new version 1.5 small RNA 3'adapter

They are apart of the new smallRNA kit or you can buy (or make them if you are happy to validate) as part of the small RNA oligo only kits from illumina.

Nik

[mnkyboy]

v1.5 sRNA 3'adapter is a new version 1.5 small RNA 3'adapter

They are apart of the new smallRNA kit or you can buy (or make them if you are happy to validate) as part of the small RNA oligo only kits from illumina.

Nik

The v1.5 sRNA 3' adapter do not come with the oligo only kits you can only get them from the complete small RNA library kits.

[dawe]

Hi

joa_ds:
However, people are discovering that even when you remove ribosomal RNA, there are several ncRNA species that are very very abundant and suck up a lot of reads. So even with ribo-minus kit, number of reads necessary to get a comprehensive coverage of the human transcriptome will be pretty high. We estimate that with polyA selected RNA-seq at 60mln reads we are getting very deep coverage of the transcriptome, so without polyA selection you probably will have generate more than 100-150 million reads to get a comprehensive transcriptome profile.

can you provide some references for these findings? i.e. about the ncRNA issue and the estimation of number of reads needed.
Thanks

d

[cmawhinney]

The 5X frag buffer in the published protocol is not the same as the 10X Ambion product. 10X product is a buffered Zinc solution and fragmentation is at 70C - metal based fragmentation method. The 5x solution is based on the Affy buffer (TrisAcetate + KOAc + MgOAc) and fragmentation is carried out at the higher temp of 94C.

Both buffers work well - we have not switched from the Ambion buffer and have made many RNAseq libraries both non directional and directional without issue.

Thanks for this info. Super helpful. How are you using the Ambion frag buffer then? Are you starting with 50 - 100ng of mRNA? Are you following Ambions protocol for 70C x 15 minutes? And in what volume?

Thanks in advance!!

[tamar]

Directional mRNA seq

Has anyone used this protocol with adaptors from the truseq small RNA kit?

[amitm]

references please

joa_ds:
... people are discovering that even when you remove ribosomal RNA, there are several ncRNA species that are very very abundant..
We estimate that with polyA selected RNA-seq at 60mln reads we are getting very deep coverage of the transcriptome, so without polyA selection you probably will have generate more than 100-150 million reads to get a comprehensive transcriptome profile.
..

hello joa_ds,
We plan to do polyA-neutral RNA-Seq on Illumina. So have been looking for information about estimating coverage/ the right 'depth' for capturing the Total RNA quantitatively (possible..?).
Your post is very informative. Would you please guide me to concerned references from where you have quoted the figures.
That would be a lot of help

thanks

[Garyron]

Directional Library from Bacterial mRNA

Hello All,
I am trying to prep directional libraries from bacterial mRNA. I want to use truseq adapters for that, but the concentrations of these adapters are different from the 1.5V adapters. Please let me know if you have tried this. Thanks

[Garyron]

Hello protist,
Could you tell me tru seq small RNA adapters can be used for directional seq of bacterial mRNA and what's their concentration?
Thanks