Oligo pool design and library
We designed each the oligonucleotide to contain a guide RNA encoding
sequence and its target sequence flanked by the correct PAM(TTTV) , in a
total length of 140 bases(Fig1.a) 12,044 guide RNA sequences were
generated targeting human genes, and 500 were generated targetingE. coli genome.
Each oligonucleotide included two constant 20-base sequences at either
end for PCR amplification; A unique 8-base barcode sequence was inserted
in each oligonucleotide.
Oligonucleotides were synthesized electrochemically on arrays (Custom
Array, GenScript). These oligonucleotides (140 bases each) were
amplified by PCR using Q5 Polymerase (NEB) and gel-purified by using a
QIAquick Purification Kit (QIAGEN). Purified PCR products were assembled
with the PUC19 vector using a NEBuidler HiFi DNA assembly kit (NEB).
Assembly reaction products were transformed into Trans1-T1 competent
cells (Transgene). Transformed cells were cultured onto Luria–Bertani
(LB) agar plate with 100 μg/ml ampicillin. The resulting number of
colonies yielded more than 60× library coverage. The total plasmids were
extracted with a Plasmid Maxiprep Kit (Biomed).
Library sequences are given in TABLE.(excel)