Homemade Elute/Prime/Fragment mix?

[eab]

Does anyone know how to duplicate the 1-step elution/priming/fragmentation step from the TruSeq mRNA sequencing protocol using homemade reagents? I have oligo(dT) beads and buffers, fragmentation buffer (Zn-based, from Ambion), and random primers. My problem is that I don't know how to mix the fragmentation buffer and random primers to make the reagent that elutes and fragments while still being compatible with downstream cDNA synthesis. Do I need to use a different fragmentation reagent?

Anyone who has used the Illumina protocol knows that it saves a good bit of time - and allows 96-well plate processing - not to have to purify the RNA out of the fragmentation buffer before proceeding with RT.

Thanks!

[upendra_35]

I am also looking forward to knowing this mix. Anybody can help with this?

[eab]

Hi upendra, I've decided to assume that it's a magnesium-based fragmentation. Whatever is in there has to be compatible with the Superscript II RT reaction (since there's no purification in between), so I don't see what else it can be. Therefore, I was planning on giving that a shot, basically by just working backwards from the known components and concentrations recommended by Invitrogen for SSII. I can let you know how that goes. If you hit on anything that works well, would you let me know?
Thanks
eab

[upendra_35]

Thanks eab. I was using TruSeq recently and came to know about this mix. But i won't be using this kit for longer as it is expensive and decide to use our own in-house protocol and for that i need to know this mix. Thanks for sharing this info.

[eab]

sure thing.
one warning: you might want to check exactly how much it will cost per library to make libraries according to the TruSeq protocol using homemade reagents. you can probably have the oligos you need synthesized at a reasonable cost, but there are a lot of different enzymes in there that cost hundreds each per vial. the savings may not be as great as you might think.

[upendra_35]

Hi eab,
We have worked out on that. The cost per library with the home-made kit is around ~$22 including RNA extraction. This is pretty less compared to TruSeq which is ~$70 excluding RNA extraction. So we cannot afford to use TruSeq kit as we need to make around 2000 libraries in near future.

[eab]

Hi upendra, I am in the same boat with you, but I came up with a very different cost number for home-made library prep. Here are the per-sample dollar costs I've calculated. Can you tell me where we differ?

$10.75 mRNA purification beads
$1.38 random hexamer primers
$5.38 superscript II RT
$6.38 RNAse H
$13.60 DNA Polymerase I
$2.20 second strand buffer
$1.26 dNTPs
$6.10 T4 DNA polymerase
$5.30 T4 polynucleotide kinase
$1.40 Klenow fragment
$4.30 Klenow fragment (3'-5' exo-)
$4.38 T4 DNA ligase
$1.03 Phusion polymerase
$5.00 Adapter oligos

$68.46 total library prep

[eab]

prelim results

Hi upendra, any luck with a homemade mix?

i've found that i can fragment RNA off oligo-dT Dynabead using a homemade tris-buffered magnesium solution. it yielded a ~110-base average fragment after 8 minutes at 94 degrees, according to bioanalyzer. looked comparable to the illumina EPF mix, though i haven't taken it all the way through library construction yet so i don't know for sure.

bottom line: it's easy to fragment mRNA off the beads with magnesium.

Thanks eab. I was using TruSeq recently and came to know about this mix. But i won't be using this kit for longer as it is expensive and decide to use our own in-house protocol and for that i need to know this mix. Thanks for sharing this info.

[eab]

Hi Upendra, I've had some nice results working backwards from the buffer components in the superscript II RT reaction, and fragmenting for 2-3 minutes at 95 degrees C. My buffer just has Tris pH 8.3, MgCl2, and KCl. I figure on adding 19.5 microliters to the oligo dT beads, fragmenting, and then adding 17 mcl of fragmented eluate to a 25 mcl RT reaction. I did not put the random hexamers in the buffer, as they could theoretically be added in a subsequent annealing step without too much extra work.

Hi eab,
We have worked out on that. The cost per library with the home-made kit is around ~$22 including RNA extraction. This is pretty less compared to TruSeq which is ~$70 excluding RNA extraction. So we cannot afford to use TruSeq kit as we need to make around 2000 libraries in near future.

[upendra_35]

Hi Eab, Thank you very much for the recipe. Let me go ahead and try it by myself and see how it goes. Will update on that......

[silin284]

superscript buffer can be used directly as fragmentation buffer

we have been using superscript3 buffer (2x) as fragmentation solution. After the one step fragmentation/elution, we add water, dntp etc to do the RT. Because the Superscript3 First-Strand buffer has the same pH and Mg2+ like the illumina fragmentation buffer (see the protocol in this one http://www.ncbi.nlm.nih.gov/pubmed/21807852). Our collaborator also noticed that some Superscript Kit comes with a Mg2+ free buffer and Mg2+ is supplied separately. In that case, all you need to do is to add the Mg2+ to the RT buffer before using it for elution/fragmentation.

[eab]

thanks silin, glad to know this is working for someone else. what do you use for primers - random hexamers? do you anneal them in a separate step after fragmentation, or do you include them in the fragmentation mix and allow them to anneal as the reactions cool?
cheers,
eab

[silin284]

for routine rna-seq, we include hexamers in the fragmentation buffer to save some time, but for special libaries we have to add other oligos with hexamer in a later step like that protocol.