Hello,
I am new to sequencing, and plan to perform genome-wide CRISPR/Cas9 screening as described in this paper - http://www.ncbi.nlm.nih.gov/pubmed/24336571
Here, they made their own amplicons by doing 1st PCR to amplify gRNA, then 2nd PCR to attach illumina adaptors and barcodes, without using a kit.
I wish to do the same, but don't know how to design my own barcodes? And what sequence do I use for the 1-9bp variable region?
F2
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT(1-9bp variable length sequence) tcttgtggaaaggacgaaacaccg
R2 CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(8bp barcode)tctactattctttcccctgcactgt
Capitals are illumina adaptors, small letters are annealing regions.
Any suggestions are welcome.
I am new to sequencing, and plan to perform genome-wide CRISPR/Cas9 screening as described in this paper - http://www.ncbi.nlm.nih.gov/pubmed/24336571
Here, they made their own amplicons by doing 1st PCR to amplify gRNA, then 2nd PCR to attach illumina adaptors and barcodes, without using a kit.
I wish to do the same, but don't know how to design my own barcodes? And what sequence do I use for the 1-9bp variable region?
F2
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT(1-9bp variable length sequence) tcttgtggaaaggacgaaacaccg
R2 CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(8bp barcode)tctactattctttcccctgcactgt
Capitals are illumina adaptors, small letters are annealing regions.
Any suggestions are welcome.
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