In vitro selection sequences for PACE evolved TALENs targeting ATM target site
Sequences from Hubbard, et al., 2015.  The specificity profiles of PACE evolved TALENs targeting ATM target sites using an in vitro selection method.  Briefly, preselection libraries of > 10^12 DNA sequences, each theoretically containing at least 10 copies of all possible DNA sequences with six or fewer mutations relative to the on-target sequence (TGAATTGGGATGCTGTTT-random spacer- TTTATTTTACTGTCTTTA), were digested with in vitro-translated TALEN. Cleaved library members harbored a free 5’ monophosphate that enabled them to be captured by adaptor ligation.  We isolated DNA fragments of length corresponding to 1.5 target sites (an intact target site and a repeated half-site up to the point of TALEN-induced DNA cleavage) by gel purification. High-throughput sequencing of TALEN-treated or control samples that survived this selection revealed the abundance of all TALEN-cleaved sequences as well as the abundance of the corresponding sequences before selection.  In the control sample, all members of the preselection library were cleaved by a restriction endonuclease (NEB Acc65I) at a constant sequence to enable their capture by adaptor ligation and isolation by gel purification.   Selections were indexed with the first 5 bp of sequence.  Thus, the first 5bp of each sequences identifies which selection condition the sequence originated from: For sequences fastq files:"ACTGT" = Canonical 20 nM, "CTGAA" = Canonical 10 nM, "TGACT" = Canonical 5 nM, "TGCAA" = Canonical 2.5 nM, "GCATT" = L3-1 20 nM, "CATGA" = L3-1 10 nM, "ATGCT" = L3-1 5 nM, "CTAGT" = L3-1 2.5 nM, "GCTAA" = L3-2 10 nM, "CAGTT" = L1-1 10 nM.   Pre-selection library sequences are in of the format “NNNNGGTACCCCACTCCGCGT”