New RNA-Seq library prep kit

epibio · 10-11-2010, 12:22 PM

Epicentre Biotechnologies announces the availability of the new ScriptSeq(TM) mRNA-Seq Library Preparation Kit (Illumina-compatible). The kit uses a unique, patented terminal-tagging technology that simplifies strand-specific tagging and the preparation of directional mRNA-Seq libraries from rRNA-depleted or poly(A) RNA.

Starting from just 50 ng of rRNA-depleted mRNA, or poly(A) RNA, the ScriptSeq kit produces adaptor-tagged libraries in about 3 hours without the need for end-repair, adaptor ligations, shearing the cDNA, or gel electrophoresis.

For more information, please see the product page.

mnkyboy · 10-14-2010, 03:39 PM

Do you have any information on how this would perform for whole transcriptome analysis and not just mRNA-seq?

epibio · 10-15-2010, 07:47 AM

The kit is actually suitable for whole-transcriptome analysis, since it uses random-primed cDNA synthesis. The choice of mRNA-Seq or WT would be determined by whether you were using poly(A)-enriched or rRNA-depleted RNA, respectively.

cldashie · 10-15-2010, 08:41 AM

Hi - I was wondering if your adapters were paired-end ready for the mRNA seq kit, and if your adapters are computable with Illumina's indexes and multiplexing pcr primers.

epibio · 10-15-2010, 04:20 PM

Yes, the adaptors are designed to enable paired-end reads. They are also suitable for multiplexing, either with our barcodes kit or with barcodes that you design.

upendra_35 · 10-15-2010, 06:13 PM

do you know when will the Ribo-zero rRNA kit will be available for plant RNA?

epibio · 10-18-2010, 07:14 AM

Answered via PM.

MaizeMaze · 12-01-2010, 11:19 AM

Can you PM me the ribo-zero for plant info too?
Thanks!

epibio · 12-02-2010, 07:35 AM

I don't think I can PM you as yet, since you're a new user. However, we expect the plant kits to be released early next year. If you're interested in beta-testing the kits, please contact me using the e-mail link in my profile.

arabidopsis · 02-24-2011, 02:03 AM

ScriptSeq kit ia supposed to produce adaptor-tagged libraries from as little as 50 ng of rRNA-depleted mRNA. My question is addressed to the the vendor (epibio?) as well as to the experienced users.
I produced some libraries using 200 ng rRNA-depleted RNA as a starting material. After 10 cycles of PCR amplification, recommended by Epicentre, DNA is barely detectable by spectrophotometry (concentration less than 50 ng/ul) What is the expected yield of double-stranded DNA (final product)? Is it normal to have such low yield, or something have gone wrong, or else my spec is just out of order?

HESmith · 02-24-2011, 07:39 AM

50 ng/ul is plenty! One ng of library is sufficient for an entire flow cell (~500 million reads on a HiSeq).

arabidopsis · 02-24-2011, 09:12 AM

Thanks HESmith!
I have no experience with those libraries; neither do other people in the lab. This would be our first RNAseq project

, thus I have been quite nervous about it

.

rRNA removal kit of Epicentre worked pretty good, by the way. qPCR shown 78,000-fold reduction of rRNA amplicon signal.

epibio · 02-24-2011, 11:13 AM

@ arabidopsis: Assuming you had 50 ul after PCR, that should be plenty, as HESmith said. For the kit QC, we use 100 ng of poly(A)+ RNA as input, and obtain around 150 ng of the cDNA library.

RCJK · 03-24-2011, 02:36 AM

@epibio: have you had any feedback from anyone using the mRNA-seq kits for 454? Are you able to point me to anywhere that would have this info? Thanks.

epibio · 03-25-2011, 08:15 AM

We have not heard from any customers using the ScriptSeq Titanium-compatible kit. Most of our development efforts have focused on the Illumina platform, but the Titanium-compatible kit is very similar to the Illumina-compatible one, with the exception of adaptor sequences, and should perform equivalently.

annaoc · 06-03-2011, 04:22 AM

Hi everyone. With the ScriptSeq mRNA-Seq Library Preparation Kit, would you recommend the MinElute or gel electrophoresis for library purification?
Has anyone had experience of primer dimers after the PCR of 10 cycles? Cutting the band via size selection from a gel would perhaps then be the better choice. However, we do realise that by using the MinElute we can avoid exposing our sample to UV and Ethidium bromide and so we will probably obtain a much purer sample from MinElute as well as it being much a much easier protocol. Any comments on this would be greatly welcomed!
Also, will our library still include the tRNA and small RNAs? Our bioanalyser run of the rRNA depleted RNA has shown that we have quite a lot of tRNA and we were wondering if they will be in the library then we can avoid them by size selection on the gel?
Many thanks for your help!

epibio · 06-06-2011, 08:09 AM

We recommend using 1.2X Ampure XP beads, if you want to get rid of the small amplicons. MinElute is not as good at doing this; you can expect ~10% adaptor-related reads with MinElute as compared to <3% with Ampure.

jagruh · 06-14-2011, 09:58 AM

epibio: Is ScriptSeq mRNA-seq library prep kit (for Illumina) strand specific? From the mechanism, it seems strand specific, but in our sequenced result there are still enormous hits to the other strand. We wonder whether these anti-transcripts are biological (in bacteria) or artifacts.
Also, The distribution of hits within one gene is very uneven. It seems the PCR artifacts and/or non-random fragmentation inference a lot. Is there any available normalization parameters or tools available for this kit? And is there any published data prepared by this kit available in SRA database?
Thank you!

arabidopsis · 09-30-2011, 06:28 AM

Dear all,

I am analyzing the result of RNAseq on Hela cells. Libraries were created by Epicentre ScriptSeq kit, and they have around 7.000.000 reads each. I mapped the reads with TopHat to both hg18 and hg19. Most of transcripts look OK, but weird thing happens with RNA genes-snRNA, snoRNAs etc. Almost all reads cover reference RNAs starting from nucleotide +5 to +10, not from the annotated 5' end, even if the total coverage is very high. Out of more than 2000 reads mapped to U1 snRNA only 2 or 3 have their 5' end identical to the gene. Have anyone experienced this? Is there some technical issue with the library construction or sequencing, or a mapping artifact?
Best,

Aleks

epibio · 09-30-2011, 09:14 AM

@arabidopsis: The ScriptSeq method is designed for mRNA-Seq, and is probably not the best fit for small RNAs. It may be partly a mapping problem as well; you could try allowing for more mismatches.

@jagruh: Sorry I missed your post--for others who are interested, the ScriptSeq method is directional; however, we don't have data published to SRA as yet.

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10-11-2010, 12:22 PM	#1
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	New RNA-Seq library prep kit Epicentre Biotechnologies announces the availability of the new ScriptSeq(TM) mRNA-Seq Library Preparation Kit (Illumina-compatible). The kit uses a unique, patented terminal-tagging technology that simplifies strand-specific tagging and the preparation of directional mRNA-Seq libraries from rRNA-depleted or poly(A) RNA. Starting from just 50 ng of rRNA-depleted mRNA, or poly(A) RNA, the ScriptSeq kit produces adaptor-tagged libraries in about 3 hours without the need for end-repair, adaptor ligations, shearing the cDNA, or gel electrophoresis. For more information, please see the product page.

02-24-2011, 09:12 AM	#12
arabidopsis Member Location: Denmark Join Date: Oct 2010 Posts: 13	Thanks HESmith! I have no experience with those libraries; neither do other people in the lab. This would be our first RNAseq project , thus I have been quite nervous about it . rRNA removal kit of Epicentre worked pretty good, by the way. qPCR shown 78,000-fold reduction of rRNA amplicon signal. Last edited by arabidopsis; 02-24-2011 at 09:17 AM.

10-14-2010, 03:39 PM	#2
mnkyboy Member Location: Seattle, WA Join Date: Mar 2009 Posts: 87	Do you have any information on how this would perform for whole transcriptome analysis and not just mRNA-seq?

10-15-2010, 07:47 AM	#3
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	The kit is actually suitable for whole-transcriptome analysis, since it uses random-primed cDNA synthesis. The choice of mRNA-Seq or WT would be determined by whether you were using poly(A)-enriched or rRNA-depleted RNA, respectively.

10-15-2010, 08:41 AM	#4
cldashie Junior Member Location: NC Join Date: Jun 2010 Posts: 2	Hi - I was wondering if your adapters were paired-end ready for the mRNA seq kit, and if your adapters are computable with Illumina's indexes and multiplexing pcr primers.

10-15-2010, 04:20 PM	#5
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	Yes, the adaptors are designed to enable paired-end reads. They are also suitable for multiplexing, either with our barcodes kit or with barcodes that you design.

10-15-2010, 06:13 PM	#6
upendra_35 Senior Member Location: USA Join Date: Apr 2010 Posts: 102	do you know when will the Ribo-zero rRNA kit will be available for plant RNA?

10-18-2010, 07:14 AM	#7
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	Answered via PM.

12-01-2010, 11:19 AM	#8
MaizeMaze Junior Member Location: USA Join Date: Dec 2010 Posts: 1	Can you PM me the ribo-zero for plant info too? Thanks!

12-02-2010, 07:35 AM	#9
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	I don't think I can PM you as yet, since you're a new user. However, we expect the plant kits to be released early next year. If you're interested in beta-testing the kits, please contact me using the e-mail link in my profile.

02-24-2011, 02:03 AM	#10
arabidopsis Member Location: Denmark Join Date: Oct 2010 Posts: 13	ScriptSeq kit ia supposed to produce adaptor-tagged libraries from as little as 50 ng of rRNA-depleted mRNA. My question is addressed to the the vendor (epibio?) as well as to the experienced users. I produced some libraries using 200 ng rRNA-depleted RNA as a starting material. After 10 cycles of PCR amplification, recommended by Epicentre, DNA is barely detectable by spectrophotometry (concentration less than 50 ng/ul) What is the expected yield of double-stranded DNA (final product)? Is it normal to have such low yield, or something have gone wrong, or else my spec is just out of order?

02-24-2011, 07:39 AM	#11
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	50 ng/ul is plenty! One ng of library is sufficient for an entire flow cell (~500 million reads on a HiSeq).

02-24-2011, 11:13 AM	#13
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	@ arabidopsis: Assuming you had 50 ul after PCR, that should be plenty, as HESmith said. For the kit QC, we use 100 ng of poly(A)+ RNA as input, and obtain around 150 ng of the cDNA library.

03-24-2011, 02:36 AM	#14
RCJK Senior Member Location: Australia Join Date: May 2009 Posts: 155	@epibio: have you had any feedback from anyone using the mRNA-seq kits for 454? Are you able to point me to anywhere that would have this info? Thanks.

03-25-2011, 08:15 AM	#15
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	We have not heard from any customers using the ScriptSeq Titanium-compatible kit. Most of our development efforts have focused on the Illumina platform, but the Titanium-compatible kit is very similar to the Illumina-compatible one, with the exception of adaptor sequences, and should perform equivalently.

06-03-2011, 04:22 AM	#16
annaoc Junior Member Location: Belfast, U.K. Join Date: May 2011 Posts: 1	Hi everyone. With the ScriptSeq mRNA-Seq Library Preparation Kit, would you recommend the MinElute or gel electrophoresis for library purification? Has anyone had experience of primer dimers after the PCR of 10 cycles? Cutting the band via size selection from a gel would perhaps then be the better choice. However, we do realise that by using the MinElute we can avoid exposing our sample to UV and Ethidium bromide and so we will probably obtain a much purer sample from MinElute as well as it being much a much easier protocol. Any comments on this would be greatly welcomed! Also, will our library still include the tRNA and small RNAs? Our bioanalyser run of the rRNA depleted RNA has shown that we have quite a lot of tRNA and we were wondering if they will be in the library then we can avoid them by size selection on the gel? Many thanks for your help!

06-06-2011, 08:09 AM	#17
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	We recommend using 1.2X Ampure XP beads, if you want to get rid of the small amplicons. MinElute is not as good at doing this; you can expect ~10% adaptor-related reads with MinElute as compared to <3% with Ampure.

06-14-2011, 09:58 AM	#18
jagruh Junior Member Location: Richmond, VA Join Date: Apr 2011 Posts: 1	epibio: Is ScriptSeq mRNA-seq library prep kit (for Illumina) strand specific? From the mechanism, it seems strand specific, but in our sequenced result there are still enormous hits to the other strand. We wonder whether these anti-transcripts are biological (in bacteria) or artifacts. Also, The distribution of hits within one gene is very uneven. It seems the PCR artifacts and/or non-random fragmentation inference a lot. Is there any available normalization parameters or tools available for this kit? And is there any published data prepared by this kit available in SRA database? Thank you!

09-30-2011, 06:28 AM	#19
arabidopsis Member Location: Denmark Join Date: Oct 2010 Posts: 13	Dear all, I am analyzing the result of RNAseq on Hela cells. Libraries were created by Epicentre ScriptSeq kit, and they have around 7.000.000 reads each. I mapped the reads with TopHat to both hg18 and hg19. Most of transcripts look OK, but weird thing happens with RNA genes-snRNA, snoRNAs etc. Almost all reads cover reference RNAs starting from nucleotide +5 to +10, not from the annotated 5' end, even if the total coverage is very high. Out of more than 2000 reads mapped to U1 snRNA only 2 or 3 have their 5' end identical to the gene. Have anyone experienced this? Is there some technical issue with the library construction or sequencing, or a mapping artifact? Best, Aleks

09-30-2011, 09:14 AM	#20
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	@arabidopsis: The ScriptSeq method is designed for mRNA-Seq, and is probably not the best fit for small RNAs. It may be partly a mapping problem as well; you could try allowing for more mismatches. @jagruh: Sorry I missed your post--for others who are interested, the ScriptSeq method is directional; however, we don't have data published to SRA as yet.