SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
SMARTer Ultra low RNA Kit Primers are found in sequencing data and Tophat questions TEFA Sample Prep / Library Generation 12 12-02-2015 07:48 AM
Library prep kit for low input RNA seq Agun Sample Prep / Library Generation 9 03-13-2014 12:26 PM
Clontech Low Input Library Prep Kit slees Sample Prep / Library Generation 3 02-11-2014 09:24 AM
SMARTer Ultra Low RNA (Clonetech, 634935) jyansouni Sample Prep / Library Generation 1 12-04-2012 10:12 AM
New low-input rRNA removal kit, ideal for FFPE samples epibio Vendor Forum 0 11-04-2010 09:55 AM

Reply
 
Thread Tools
Old 03-19-2014, 07:40 AM   #1
vanillasky
Member
 
Location: Europe

Join Date: Mar 2014
Posts: 41
Default SMarter Low RNA Input Kit

So I have used the SMarter Low RNA Input Kit and after illumina sequencing with an NEB kit we found out that the sequencing pretty much failed. Although I've contacted the company regarding the results I have yet to receive any tech support. The following attachment shows the dscDNA output as measured with a High Sensitivity Kit. The pattern resembles their control outcome described in the manual. Any input would help me figure out where things went wrong.
Attached Files
File Type: pdf chip result.pdf (65.4 KB, 69 views)
vanillasky is offline   Reply With Quote
Old 03-19-2014, 10:53 AM   #2
Simone78
Senior Member
 
Location: Bonn (Germany)

Join Date: Oct 2010
Posts: 199
Default

Why don´t you try Smart-seq2+Nextera instead? It would be much cheaper...at least up to the pre-amplification step! Your RNA seems to be degraded, do you have a positive control ? You could do RNA extraction from bulk, take 1 ng and do 12-14 cycles PCR. You should get plenty of cDNA.
Simone78 is offline   Reply With Quote
Old 03-19-2014, 12:16 PM   #3
vanillasky
Member
 
Location: Europe

Join Date: Mar 2014
Posts: 41
Default

I picked this kit because I am working with low amounts of degraded RNA and this kit was specifically developed for low input pg-ng and for samples that are degraded. The RNA was extracted from a marine sediment. What do you mean by 12-14 cycles of PCR?
vanillasky is offline   Reply With Quote
Old 03-20-2014, 01:38 AM   #4
Simone78
Senior Member
 
Location: Bonn (Germany)

Join Date: Oct 2010
Posts: 199
Default

I mean that after RT you would need 12-14 cycles of PCR to get enough DNA that you can continue with library prep (and check on the Bioanalyzer).
Simone78 is offline   Reply With Quote
Old 11-23-2015, 02:48 PM   #5
efrainrib
Junior Member
 
Location: NYC

Join Date: Nov 2015
Posts: 3
Default

I've been using the SMARTER total RNA-seq kit to make libraries from nuclear RNA, ranging from 1-10ng. Honestly when I go under 1ng my libraries are not there. However, from 1-10 ng we have made over 100 libraries that look like the traces provided at the end of the clontech manual. We've probably tried upwards of 150... so you can do the math and see the throughput. We've sequenced it already and are working on the analysis of the data, and from our end everything about the kit looks as advertised. would be happy to answer any more questions in depth if youre still doing this stuff
efrainrib is offline   Reply With Quote
Old 12-01-2015, 05:09 AM   #6
NextGenSeq
Senior Member
 
Location: USA

Join Date: Apr 2009
Posts: 482
Default

We use the NuGen RNA-Seq V2. If your RNA is degraded use the FFPE RNA-Seq V2.

It works with anything.
NextGenSeq 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 04:04 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO