Hello everyone,
I have been given a little funding for an undergraduate research project and decided on performing differential gene expression through RNA-Seq on the two mouse models I have access to. After designing my experiment, and budgeting for a couple of lanes on the Illumina HiSeq, I was surprised to learn that the university core facility charges a per sample fee to prepare the libraries, and those fees make me go over my budget.
Because I do have access to a few different kits and reagents in the lab I'm wondering whether I can adapt the TruSeq protocol to fit my needs. My objective in developing a modified protocol is to:
1) Decrease per sample costs of library prep in order to afford a higher number of replicates;
2) Limit the introduction of bias (lane, batch, pcr, etc.).
After reading a few papers and many posts on seqanswers.com I came up with the following protocol. Will this work? What kinds of problems will I encounter if I prepare my samples in this manner? Any insights will be greatly appreciated.
1. Isolate mRNA (Invitrogen Dynabeads)
2. Verify purity
3. Fragment mRNA (Ambion Fragmentation kit)
4. Verify size range
5. Repair ends (T4 polynucleotide kinase, ATP)
6. Ligate 6-bp barcode to 5' end (NEB T4 RNA Ligase, Custom made barcodes)
7. Verify ligation, purity
8. Pool samples in equimolar concentrations
9. Submit as one single sample to core facility for remaining steps of TruSeq library prep
I have been given a little funding for an undergraduate research project and decided on performing differential gene expression through RNA-Seq on the two mouse models I have access to. After designing my experiment, and budgeting for a couple of lanes on the Illumina HiSeq, I was surprised to learn that the university core facility charges a per sample fee to prepare the libraries, and those fees make me go over my budget.
Because I do have access to a few different kits and reagents in the lab I'm wondering whether I can adapt the TruSeq protocol to fit my needs. My objective in developing a modified protocol is to:
1) Decrease per sample costs of library prep in order to afford a higher number of replicates;
2) Limit the introduction of bias (lane, batch, pcr, etc.).
After reading a few papers and many posts on seqanswers.com I came up with the following protocol. Will this work? What kinds of problems will I encounter if I prepare my samples in this manner? Any insights will be greatly appreciated.
1. Isolate mRNA (Invitrogen Dynabeads)
2. Verify purity
3. Fragment mRNA (Ambion Fragmentation kit)
4. Verify size range
5. Repair ends (T4 polynucleotide kinase, ATP)
6. Ligate 6-bp barcode to 5' end (NEB T4 RNA Ligase, Custom made barcodes)
7. Verify ligation, purity
8. Pool samples in equimolar concentrations
9. Submit as one single sample to core facility for remaining steps of TruSeq library prep
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