GSM1544324: 2d-con_S12_L001_R1_001_AF_SOL_551; synthetic construct; OTHER
Cas9 in vitro assays and sequencing: Cas9 in vitro assays were performed in 10 ul reactions with Cas9 protein pre-incubated with gRNA, 1X NEB ThermoPol Taq buffer, dH2O, and random variant target library. Following incubation at 37° C, reactions were stopped by flash freezing in dry ice, addition of 200 ul of proteinase K buffer (0.1M NaCl, 0.1M TRIS pH 8.4, 0.1M EDTA, 1% SDS), addition of 20 ul of proteinase K (20 mg/ml), and incubation at 3 hours at 60° C. After adding 190 ul TE, 1 ul 1.5 ug of GlycoBlue, 2.5ug of yeast tRNA, and 40 ul of saturated ammonium acetate samples were extracted with phenol/ chloroform (1:1) and chloroform, ethanol precipitated, and resuspended in 10 ul of TE. PCR for a total of 26 cycles was used to amplify the retained sequences and prepare for sequencing. This included a first round (20 cycles) with short primers AF-KLA-136 and AF-KLA-140, and a second round (6 cycles) with longer primers bearing index sequences and flow cell anchors for sequencing and subsequent analysis (Table S1-2). All libraries were sequenced using the 50 cycle Illumina Miseq (100 cycle single-read) platforms. A list of experiments, experimental conditions, sequencing run IDs, and SRA accession numbers is reported in the supplemental material (Table S2). Samples that are controls are either with 0 Cas9 or have Cas9 and with a 0 time point. Generation of random variant target library: The random variant target library was generated using the pHRL-TK vector (Promega). Customized variant primers for the target library were extended and cloned into the NotI and Acc65I digested pHRL-TK vector (see Generation of random variant target library). The variant sequence was designed to be 35 nt (6 nt flanking on each side of a 23 nt target sequence). The 12 nt of flanking sequence and the N in the PAM sequence of the target were synthesized with custom mixes that were made up of 25% adenine (A), 25% thymine (T), 25% cytosine (C), or 25% guanine (G). The 22 bps sequence of unc-22A and ps4 were synthesized with custom mixes designed to produce a 10% variant rate at each position (3.3% of each variant base), although, the ps4 library has a unexpectedly high variant rate at two positions.The initial oligonucleotide library preparations were extended using NEB ThermoPol Taq polymerase and primer AF-KLA-87. Reaction tubes with 100 pM of each primer in 25 ul dH2O and 2.5 ul of 10X Taq ThermoPol buffer (20 mM Tris-HCl, 10 mM KCl, 2 mM MgSO4, 10mM (NH4)2SO4 , 0.1% Triton® X-100, pH 8.8) is put in a beaker of boiling water for 5 minutes and removed from heat and allowed to cool at 45° C. The primer mix is then diluted by adding 5ul of 25 mM dNTPs, 210 ul of 1X Taq buffer, and 10 ul of NEB Taq polymerase. Extension is then allowed for 15 minutes at 72° C, with extension stopped by adding 180 ul of (1.9 NH4OAc, 19 mM EDTA, 0.38% SDS), and 1.5 ug of GlycoBlue. Following extractions with phenol/chloroform (1:1) and chloroform, and ethanol precipitation, the resulting material was resuspsended in 40 ul of TE, digested with NEB NotI+Acc65I purified following electrophoresis on a 1.5% agarose gel, and ligated into the NotI and AccI65 digested recipient vector using NEB Quick ligation. Following transformation of library efficiency DH5α cells (Invitrogen), plating on pre-warmed 15 cm plates, colonies were grown overnight at 37° C then moved and incubated at 30° C for 24 hrs. Approximately 5000 colonies were scraped off of agar plates into 10 mls of 2XTY. The 10 mls of cells were pelleted and prepped using Qiagen Midi plasmid prep kit.