Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Effects of total RNA fragmentation on small RNAs

    Hello,
    I want to sequence enriched RNA (it will include both mRNAs and bacterial small non-coding RNAs (<200 nt)). I would like to fragment long RNAs to ~100-200 nt fragments before library preparation however I am afraid that small RNAs will be fragmented too and I will lose it after cutting required length RNAs from the gel (I am planning to cut 150-350 bp fragments already with adapters). My fragmentation buffer is 10 mM Tris-HCl (pH 7.4) and 10 mM ZnCl2, reaction conditions: 75 °C for 2.5 min.
    RNAs I want to sequence are not abundant - every loss of RNA sample should be avoided!
    Maybe someone has experience with the fragmentation of total RNA? Does it affect small RNAs, is it severely fragmented? Would it be better for me to prepare different libraries - one for small RNAs, another - for long RNAs?
    Thank you for your time and answer!

  • #2
    I assume chemical fragmentation will affect the small fragments to the same degree as the big ones.
    If you have an opportunity to sonicate samples? Small fragments are pretty safe from shearing.

    Another way to achieve that might be size selection (SPRI-beads) and separate fragmentation of the large fragments. Obviously, this will entail losses and add complexity.

    You might want to have a look at the Biorad Sequoia and Diagenode D-plex library prep kits for both short and long RNAs.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Recent Advances in Sequencing Analysis Tools
      by seqadmin


      The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...
      05-06-2024, 07:48 AM
    • seqadmin
      Essential Discoveries and Tools in Epitranscriptomics
      by seqadmin




      The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...
      04-22-2024, 07:01 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, Today, 02:46 PM
    0 responses
    10 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 05-07-2024, 06:57 AM
    0 responses
    13 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 05-06-2024, 07:17 AM
    0 responses
    16 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 05-02-2024, 08:06 AM
    0 responses
    23 views
    0 likes
    Last Post seqadmin  
    Working...
    X