GSM1574086: pUC19; Cloning vector pUC19; OTHER
Integration reactions were performed with Cas1-Cas2, protospacer, and plasmid.   The integration reaction was performed with 75 nM Cas1   Cas2, 200 nM  protospacer and 7.5 nM pCRISPR or pUC19 in 20 mM HEPES, pH 7.5, 25 mM  KCl, 10 mM MgCl2, 10% DMSO and 1 mM DTT. The DNAs were isolated by phenol-chloroform extraction and ethanol    precipitation. The excess protospacers were removed using 100K MWCO    Amicon Ultra-0.5 mL centrifugal filters. The resulting integration    products were digested into smaller DNA fragments using dsDNA    Fragmentase (New England Biolabs) for 75 min at 37   C and quenched at    65   C for 15 min. Fragments were end repaired using T4 DNA Polymerase    (NEB), Klenow (NEB) and T4 PNK (NEB) and A-tailed with Klenow exo (3      to 5   exo minus) (NEB). Adapters were ligated onto fragments using T4    DNA ligase (NEB) and cDNA libraries were amplified by PCR using    Phusion (NEB). Integration products were fragmented, end-repaired, adapter ligated, PCR amplified, and analyzed by Illumina sequencing. Libraries were sequenced on an Illumina HiSeq2500 on rapid run mode.
GEO
GSM1574086