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Old 06-09-2011, 08:29 AM   #1
Garyron
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Default mRNA from Bacterial sample for trueseq

Hi All,
Has anyone made directional libraries from bacterial mRNA using Truseq small RNA sample prep kit?. How does the mRNA prep look on the bioanalyser? what fragmentation time is typically used for bacterial mRNA?
Thanks
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Old 06-16-2011, 09:16 PM   #2
ScottC
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Hi,

Are you trying to make a small RNA library or an mRNA library?
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Old 06-22-2011, 04:04 PM   #3
Garyron
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Iam trying to make mRNA library.
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Old 06-22-2011, 04:54 PM   #4
ScottC
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Hi,

Do you specifically need to use the small RNA kit? We regularly prepare RNA-seq libraries for bacterial transcriptomics using the TruSeq DNA prep kit with a few modifications if we want strand specific RNA sequencing.

You can get the general idea from this paper:
http://www.ncbi.nlm.nih.gov/pubmed/19620212

Very basically, convert your RNA to cDNA, do a second strand synthesis incorporating UTP and use the double stranded material in a standard genomic DNA library prep. Immediately before PCR amplification, carry out a UTP digestion using UNG (uracil glycosylase). Amplify as normal and sequence your library.


Cheers,

Scott.
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Old 07-20-2011, 05:31 AM   #5
mgolo
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Default Analysis of strand specific data

Quote:
Originally Posted by ScottC View Post
Hi,

Do you specifically need to use the small RNA kit? We regularly prepare RNA-seq libraries for bacterial transcriptomics using the TruSeq DNA prep kit with a few modifications if we want strand specific RNA sequencing.

You can get the general idea from this paper:
http://www.ncbi.nlm.nih.gov/pubmed/19620212

Very basically, convert your RNA to cDNA, do a second strand synthesis incorporating UTP and use the double stranded material in a standard genomic DNA library prep. Immediately before PCR amplification, carry out a UTP digestion using UNG (uracil glycosylase). Amplify as normal and sequence your library.


Cheers,

Scott.
Hi!

@Scott: I have done strand-specific libraries of a bacterial transcriptome using the small RNA kit, with modifications. Basically we only paired-end sequenced the first strand.

Now i have to analyze the data and i'm going a bit crazy about it I'm using bowtie to map the reads to the genome, then i convert the sam files to bam, and then create pileup files.

My question: Am i retaining the strand info? If yes, how can i see it? If no, what should i do to retain the strand info?

Thanks in advance for your help!

Maria
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Old 07-20-2011, 05:37 AM   #6
mgolo
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Quote:
Originally Posted by Garyron View Post
Hi All,
Has anyone made directional libraries from bacterial mRNA using Truseq small RNA sample prep kit?. How does the mRNA prep look on the bioanalyser? what fragmentation time is typically used for bacterial mRNA?
Thanks
Hi again!

@Garyron: I have done that On the bioanalyzer the libraries look like the ones prepared with the TruSeq RNA kit.

I fragmented the ribosomal-depleted RNA for 8 minutes, as in the TruSeq RNA kit.

Anyway, the suggestion from Scott might be a good solution for you.

Good luck

Maria
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Old 08-11-2011, 11:29 AM   #7
Garyron
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Default libarary prep from small RNA kit illumna

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Originally Posted by mgolo View Post
Hi again!

@Garyron: I have done that On the bioanalyzer the libraries look like the ones prepared with the TruSeq RNA kit.

I fragmented the ribosomal-depleted RNA for 8 minutes, as in the TruSeq RNA kit.

Anyway, the suggestion from Scott might be a good solution for you.

Good luck

Maria
Hi Maria,
Thanks for your reply! mRNA enrichment is challenging, which kit/ protocol was a success for you. Also what was the size of your library in bps?
After fragmentation of mRNA did you use the ethanol precipitation followed by PNk trtment followed by column purification before proceeding to Adapter ligation?
Thanks in advance
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Old 08-15-2011, 01:27 AM   #8
mgolo
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Quote:
Originally Posted by Garyron View Post
Hi Maria,
Thanks for your reply! mRNA enrichment is challenging, which kit/ protocol was a success for you. Also what was the size of your library in bps?
After fragmentation of mRNA did you use the ethanol precipitation followed by PNk trtment followed by column purification before proceeding to Adapter ligation?
Thanks in advance
I used the TruSeq Small RNA Sample Prep Protocol. And, yes, after the fragmentation you need to treat with PNK, else the adapters might not ligate correctly afterwards. You can do a EtOH precipitation or column purification after PNK treatment. The average size of my libraries was about 200-250 bp.

Cheers

Maria
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Old 08-15-2011, 05:20 AM   #9
Garyron
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Hi Maria,
Thanks for your reply
It look like you have made small RNA libraries using the truseq kit, Did you make mRNA libraries with bacterial sample.. If yes then you must have used the mRNA enrichment kit which comes separate, right?
Thanks again
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Old 08-16-2011, 12:42 AM   #10
mgolo
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Quote:
Originally Posted by Garyron View Post
Hi Maria,
Thanks for your reply
It look like you have made small RNA libraries using the truseq kit, Did you make mRNA libraries with bacterial sample.. If yes then you must have used the mRNA enrichment kit which comes separate, right?
Thanks again
You can use different mRNA enrichment kits, there are many. i used the MICROBExpress kit from AMBION
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