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Using Covaris S2 for RNA shearing

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  • Hamid
    replied
    Physical shearing is better since it is concentration independent, isothermal, easily controllable as compared to chemical methods, and you don't have to deal with the stringent cleanup methods required after chemical/heat fragmentation.

    Thank you.

    Leave a comment:


  • aleferna
    replied
    Originally posted by ETHANol View Post
    I've jsut stated using the Covaris for chromatin fragmentation and so far I'm very impressed with the results. The reproducibility between samples is impressive.

    What I am wondering is what are the advantages/disavantages of using the Covaris vs. chemical methods for shearing RNA for RNA-seq library preparation?
    never got a answer for this, so what is better Covaris or the chemical/heat fragmentation? ?

    Leave a comment:


  • ETHANol
    replied
    I've jsut stated using the Covaris for chromatin fragmentation and so far I'm very impressed with the results. The reproducibility between samples is impressive.

    What I am wondering is what are the advantages/disavantages of using the Covaris vs. chemical methods for shearing RNA for RNA-seq library preparation?

    Leave a comment:


  • RCJK
    replied
    Thanks Kartik!

    Leave a comment:


  • kshankar
    replied
    RCJK
    We use the following conditions for mRNA, 120 μl volume, 10% DC, 5 intensity, 200 cpb, 7.5 min and get highly reproducible 150 bp fragmentation peaks. We are using these for Illumina libraries. If you want longer length just shorten the time. In our hands, 30 sec fragmentation gives ~ 500 bp peak, 60 sec ~ 400 bp, 180 sec ~ 200 bp fragments. hope that helps.
    -Kartik

    Leave a comment:


  • RCJK
    replied
    I just thought I'd give this thread a bump and see if there are any updates regarding using Covaris for RNA shearing, in particular for 454 libraries? We've recently obtained a Covaris and I'm hoping to try it out instead of using Roche's fragmentation procedure. Thanks!

    Leave a comment:


  • jaywang
    replied
    What is the 25bp peak in the RNA profiles?

    It looks like that there is always a small peak around 25bps. Are they mature microRNAs?
    Originally posted by Hamid View Post
    the Covaris AFA technology is very capable of shearing total RNA, as well as mRNA. We have quite a few customers who are actively using our technology in RNA-seq library preparation for SOLiD, as as Illumina platforms.
    I have attached a sample data kindly provided by a few of your customers.

    Also please follow the link http://covarisinc.com/supported-protocols.html for our 2kb, 3kb, and 5kb DNA fragmentation protocols, as we as gel and BioAnalyzer data.

    Thank you

    Hamid

    Leave a comment:


  • 04BCH022
    replied
    Originally posted by Hamid View Post
    Hi Shurjo,

    We have a few customer who have tried a 2-25 minute time course of the setting below to shear RNA:
    10%dc,5i, 1000cpb.

    A customer generated an average shearing size of 400-500b only after a 2 minute treatment using a concentration of less that 100ng/ul. My suggestion would be to use the setting above and carryout an initial test time course of 2-25 minutes depending on the concentration of the RNA sample you will ultimately shear.

    Thank you

    hamid

    Hello Hamid,

    I regularly use the covaris to shear RNA and get a nice peak at around 200bp. I am trying different settings to get that peak at 400-500bp. Are you aware of any settings which kind of shift the "triangle" further away from the marker peak (on the agilent) so that the "triangle's" peak is around 400-500bp.
    I have ran a bunch of different sets with varying intensities, time C/B, DC but havent yet found the correct configuration.

    Thank You.

    Regards.

    Leave a comment:


  • Hamid
    replied
    the Covaris AFA technology is very capable of shearing total RNA, as well as mRNA. We have quite a few customers who are actively using our technology in RNA-seq library preparation for SOLiD, as as Illumina platforms.
    I have attached a sample data kindly provided by a few of your customers.

    Also please follow the link http://covarisinc.com/supported-protocols.html for our 2kb, 3kb, and 5kb DNA fragmentation protocols, as we as gel and BioAnalyzer data.

    Thank you

    Hamid
    Attached Files

    Leave a comment:


  • josdegraaf
    replied
    Originally posted by kshankar View Post
    This is the only place I have been able to find some starting conditions for RNA fragmentation using the Covaris S2. Using the conditions that Shurjo mentioned (and some longer times, ie upto 3 mins), we are getting 100-500 range centered around 200 bp. What is the ideal range and size for fragmented RNA for Illumina mRNA-seq? Also, Does anyone know if Covaris has come out with specific settings?

    thanks for your help
    --K
    Last time I spoke with them not..

    Leave a comment:


  • kshankar
    replied
    Covaris S2 polyA RNA fragmentation conditions

    This is the only place I have been able to find some starting conditions for RNA fragmentation using the Covaris S2. Using the conditions that Shurjo mentioned (and some longer times, ie upto 3 mins), we are getting 100-500 range centered around 200 bp. What is the ideal range and size for fragmented RNA for Illumina mRNA-seq? Also, Does anyone know if Covaris has come out with specific settings?

    thanks for your help
    --K

    Leave a comment:


  • ik76
    replied
    Hello,
    I was just wandering whether anybody have tried fragmenting total RNA using Covaris ( as a replacement for the fargmentation in zink solution recommnded in the Illumina mRNA sample prep protocol). If so, what was the starting concentration and Covaris conditions? Did you have problems with RNA degrading ?
    if you have started with purified mRNA, how did you measure the conc ? Will just using Agilent Pico chip work ?

    Leave a comment:


  • nextgen
    replied
    I used 500bp range as this right range for 454 (some mRNA transcripts are in that range.) You can easily get smaller size. But for random or end-to-end cDNa synthesis, 500bp is better.

    Leave a comment:


  • nextgen
    replied
    Covaris works great for RNA shearing. You can get a very tight range. Just play around with it.

    Leave a comment:


  • shurjo
    replied
    Hi Hamid,

    Thanks for the info. These days I routinely use:

    10% DC, 5 Intensity, 200 cpb and 75 seconds

    for 150ng of polyA RNA in a 120ul volume of H2O in a 6x16mm tube. This gives a nice tight distribution around 200bp every time.

    Regards,

    Shurjo

    Leave a comment:

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