Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • directional mRNA-Seq Sample Preparation

    Hello,

    I want to start to work on directional mRNA-Seq Sample Preparation.
    I would like to know if anybody has experience with the Illumina protocol -directional mRNA-Seq Sample Preparation- and/or the protocol from epicentre -ScriptSeq™ mRNA-Seq Library Preparation Kit-.
    In your experience which one gives you the best result.
    Any suggestions are very welcome.
    thanks!

  • #2
    Hi Claire1,
    Have you decided on the kit , I just saw your post and illumina's directional protocol needs items part # from two kits as well as a fragmentation buffer but it works very well.
    Bests

    Comment


    • #3
      The Illumina protocol using multiple kits is expensive and I found the Epicentre protocol to be annoying for small sample numbers and not scalable for larger sample numbers. The Epicentre method has one long t-cycling program with wait times, pauses, + 1 ul to every tube multiple times. It is quick though (I could do libraries in one day) but I found painful.

      I now use the Illumina mRNA TruSeq kit with modifications and it works very well.

      I take the sample through the TruSeq kit per protocol through 1st strand. After 1st strand I clean up the sample with Zymo's RNA C&C -5 to remove dNTPs. I then do 2nd strand with a dNTP mix with dUTP (U, C, G, A) and NEB's dNTP-free 2nd Strand reaction buffer and 2nd Strand RNA-seq module. The resultant cDNA is taken back into the TruSeq kit until right before PCR. At this point you can treat the sample with USER and then go into PCR, or go directly into PCR (The kit taq looks to choke on dUTP strand, USER step likely not needed). This has been used on 100+ samples in my lab and works well.

      Comment


      • #4
        Hello,
        thanks for your replies!

        I decided for the kit from epicentre. Before the Sample Prep I was doing a rRNA removal with the Ribo-Zero rRNA Removal Kit. Both protocols were easy to follow, fast and were working fine. Indeed the steps to pipete 1ul individually is not setted up for larger sample numbers, but this will be not a problem for us. Now we are waiting for the sequencing results.

        Very interesting your modified mRNA TruSeq protocol. With USER do you mean UNG treatment ?

        Thanks!

        Comment

        Latest Articles

        Collapse

        • seqadmin
          Strategies for Sequencing Challenging Samples
          by seqadmin


          Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
          03-22-2024, 06:39 AM
        • seqadmin
          Techniques and Challenges in Conservation Genomics
          by seqadmin



          The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

          Avian Conservation
          Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
          03-08-2024, 10:41 AM

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by seqadmin, Yesterday, 06:37 PM
        0 responses
        10 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        9 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-22-2024, 10:03 AM
        0 responses
        51 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-21-2024, 07:32 AM
        0 responses
        67 views
        0 likes
        Last Post seqadmin  
        Working...
        X