RNA Fragmentation

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Mol.biology // Next-generation sequencing

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Содержание


  • check, that you have:
» PCR thermal cycler with 70°C-hold program;
» 4°C centrifuge;
» pump;
» 5min timer;
» Stratagene cooler at -20°C
» buffers:
10X Fragmentation Reagent2µl
Glycogen, 10µg/µl2µl
Stop Buffer2µl
AcONa, 3M, pH 5.22µl
H2O20µl
EB10µl
EtOH, 100%60µl
EtOH, 70%300µl



[править] Protocol / Ambion

  • (preliminary) set PCR thermal cycler at 70°C

  1. mix in a thin-wall 200µl tube[1]:
    mRNA? µl
    10X Fragmentation Reagent2µl
    H2Oup to 20µl
  2. mix and spin down;
  3. put the tube in the preheated (70°C) PCR block for 5-15 min[2];
  4. take the tube out of the block by hand, place it on ice and add 2µl of Stop Buffer;
  5. transfer the solution to a 1.5ml tube for Ethanol precipitation:
    sheared RNA20µl
    AcONa, 3M, pH 5.22µl
    Glycogen, 10µg/µl[3]2µl[4]
    EtOH, 100%60µl
  6. mix and incubate at -20°C (in Stratagene cooler) for at least 30 min;
  7. centrifugation 14-27krpm, 4°C, 20 min;
  8. remove Ethanol;
  9. add 300µl of 70% EtOH;
  10. centrifugation 14krpm, 5 min;
  11. remove Ethanol;
  12. air dry the pellet for 5 min;
  13. resuspend in 10µl of EB



[править] Protocol / Illumina

Illumina protocol has two different points:

» 5x Fragmentation Buffer (Illumina, Cat#RS-100-0801) is used is used (4µl of 5x buffer for 20µl reaction)
» shearing performed at 94°C for exactly 5 min



[править] Solutions

[править] 10X Fragmentation Reagent

buffered zinc solution, Ambion, Cat#AM8740, 200µl
store at room temperature


[править] Stop solution

store at +4°C

conc.stock500µl
EDTA, pH 8.00.2M0.5M200µl
H2O mQ300µl




[править] Notes

  • protocol is for shearing of RNA to ~200nt fragments.


NB! chemical fragmentation does not depends on "previous degradation". It adds additional breaks in any case. In this respect it is more dangerous, than synthesis of "long cDNA" and US-shearing, because later is highly resistant to "overshearing".



[править] Open questions

  • in the "RNA Fragmentation" protocol on kreatech.com "10X Fragmentation Reagent" is: 100mM ZnCl2 in 100mM Tris-HCl pH7. It is worth to compare it with the commercial buffer on total RNA.




  1. up to 50µg of RNA may be sheared in 20µl. If necessary (diluted RNA sample or large amount of RNA), volume of shearing reaction in one tube may be increased up to 50µl (up to 125µg of RNA).
  2. 5min fragmentation for 200nt mean size in
    Vivancos AP, Gu"ell M, Dohm JC, Serrano L, Himmelbauer H. Strand-specific deep sequencing of the transcriptome. Genome Res. 2010 Jul;20(7):989-99.
    protocol for ligation-bases strand-specific RNA-Seq library preparation
  3. it is not necessary to add Glycogen, if amount of RNA is more than 1µg
  4. amount of Glycogen is the same for any volume of Ethanol precipitation reaction


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