SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Unexpected 425bp band un Truseq stranded mRNA library prep Amcb1 Sample Prep / Library Generation 5 06-07-2017 11:16 AM
Adapter titration in TruSeq stranded mRNA sample preparation chicken Illumina/Solexa 1 11-11-2016 01:18 PM
End repair of TruSeq RNA and TruSeq mRNA stranded ykgenome Sample Prep / Library Generation 2 05-15-2014 12:01 AM
silly question r.e. TruSeq stranded mRNA sweetph3 Illumina/Solexa 2 03-21-2014 01:20 PM
3'End polyA artefact in illumina TruSeq Small RNA Seq proteomania RNA Sequencing 1 10-16-2012 05:51 AM

Reply
 
Thread Tools
Old 07-11-2017, 10:45 AM   #1
Chinanicole
Junior Member
 
Location: Philadelphia

Join Date: Jun 2017
Posts: 8
Default Save truseq stranded mRNA polyA(-) supernatant for small RNAseq?

Has anyone tried to save and sequence the polyA(-) supernatant from Illumina's TruSeq stranded mRNA library prep kit? If so, any advice or insight into things to consider using the supernatant - ie: would I do another phenol extraction or something to pellet out the polyA(-) RNA then suspend in H2O?

I'm interested in small RNAs (miRNA, piRNA) in particular so I assume I'd need to clean up the supernatant on a gel to size select for 20-30 nt or maybe use another rRNA depletion to clean up the samples.

Thanks!

This kit:
https://www.illumina.com/products/by...nded-mrna.html
Chinanicole is offline   Reply With Quote
Old 07-11-2017, 11:14 PM   #2
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 1,080
Default

It is a good idea to try. Simplest way would be to dilute the bead supernatant to reduce salts concentration coming from binding buffer and then use a Zymo RNA Clean & Concentrator column which gives options to purify the required size range including small RNA for follow up application.
nucacidhunter is offline   Reply With Quote
Old 07-13-2017, 11:34 AM   #3
Chinanicole
Junior Member
 
Location: Philadelphia

Join Date: Jun 2017
Posts: 8
Default

I have a bunch of Qiagen miRNeasy columns as well (> 20 nt) - any reason to use the Zymo over the Qiagen?

Thanks!
Chinanicole is offline   Reply With Quote
Old 07-13-2017, 05:25 PM   #4
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 1,080
Default

Zymo kit is designed for purification and elution volume can be as low as 5 ul. miRNeasy is for RNA extraction from tissue and cells and has to be modified for purification with risk of loosing material.
nucacidhunter is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 12:51 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO