I am looking to do a project using the yeast KO collection. In the collection are over 6000 deletion strains, with a unique 20mer barcode in each KO. I am planning to use HIseq, and I know I can build the adapters and Illumina barcodes into the PCR primer, because the amplicon length will be the same for each (~120bp). The concern is that of the 120 bases, only the 20mer will be a unique sequence, and a colleague said I should make a 10bp linker in front of the area to be sequenced to help increase library complexity. Someone else said that phiX spikeins at 30% would be sufficient. I am just confused as to what I should do at this point. Hopefully someone has done this and can help me out. I appreciate any help I can get.
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1) have NNNN sequences to introduce diversity for the first 6 bp or so that is required
2) design staggered primers so that a part of your read will start on a different base (have offset versions that start extension @ 1, 2, 3, 4, 5 bp for example) -
Hi dear
I am doing the same project and we are about to use the 20mers as well.
I also have another question. When I add the inhibitory compound to my pool library in liquid pool, I can start with the OD of 0.06 - 0.08. When I go higher , my compound can not inhibit the growth well. I was wondering if you also start this OD of cells of a certain cell density. I have got the diploid library.
Which paper you are following?Comment
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