Publication protocol
"The single-stranded oligonucleotides were synthesized by the Oligo Library Synthesis (OLS) platform (Agilent Technologies, Santa Clara, CA) in either the Watson or Crick orientation in order to minimize each oligonucleotide’s frequency of adenine bases. From the oligonucleotide pool generated by OLS, we generated edit-directing plasmid pools via a ligation-mediated cloning scheme described below (graphically summarized in Supplementary Figure 1). Oligonucleotides were amplified on an AriaMx real-time PCR system (Agilent Technologies, Santa Clara, CA), using the KAPA Library Amplification kit (Kapa Biosystems, Wilmington, MA). The amplification primers were designed to introduce an EagI cut site into the 3’ end of the amplification product (Supplementary Table 7, primers named OLS Library Amplification F and R). Reactions were stopped during linear amplification, and the amplified library was then purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany), digested with BstEII-HF and EagI-HF (New England Biolab, Ipswich, MA), and purified again.
The amplified library was cloned into pLK88, a version of pLK78 modified to include the BstEII and SphI sites. pLK88 was isolated with a QIAGEN Plasmid Plus Maxiprep kit (Qiagen, Hilden, Germany) from 200 milliliters of Escherichia coli culture. 20 micrograms of plasmid was then digested by BstEII-HF and EagI-HF, treated with Shrimp Alkaline Phosphatase (New England Biolabs, Ipswich, MA), and purified with the Qiagen PCR purification kit. We tested two ligation reactions: 1 microgram of digested vector was ligated with either 100 nanograms or 800 nanograms of the digested insert, with 4 microliters of T4 DNA Ligase M0202M (New England Biolabs, Ipswich, MA), in an 800 or 200 microliter reaction, respectively, at room temperature for 10 minutes. Concurrently, we ran negative control ligations lacking the insert DNA. Ligation reactions were stopped on ice. To test ligation efficiency, 0.5 microliter of each ligation was transformed into OneShot chemically competent E. coli (Thermo Fisher Scientific, Waltham, MA). Both ligations were successful, so we proceeded to pool the ligations and concentrated them with the DNA Clean and Concentrator 25 kit (Zymo Research, Irvine, CA), eluting in 25 microliters. Then, we transformed 10 microliters of concentrated ligation product into 10 reactions of Supreme DUO electro-competent E. coli cells (Lucigen, Middleton, WI) with a Bio-Rad Micropulser (Bio-Rad Laboratories, Inc., Hercules, CA) and 0.1 cm E. coli Pulser cuvettes (Bio-Rad Laboratories, Inc., Herculues, CA). After one hour of rescue growth, cells were transferred to 200 milliliters of LB medium with 100 μg/mL Ampicillin (Sigma-Aldrich Corporation, St. Louis, MO), and grown overnight. From serial dilutions plated after the transformation, we estimated that approximately 700,000 E. coli cells were transformed. Plasmids were maxiprepped from 150 milliliters of culture.
Next, we cloned in the remaining gRNA structural region and terminator between the gRNA targeting sequence and repair template of the first-step cloning product, while adding a 12-nt barcode adjacent to the repair template (Supplementary Figure 1). The cloning insert also included a Kan-resistance gene to facilitate enrichment for the correct cloning product. The insert sequence was Pfu Ultra II PCR-amplified from pLK89 (the amplification primers used are named Insert Amplification in Supplementary Table 7). The barcode was introduced during the insert PCR amplification by use of mixed bases in the synthesis of the reverse primer. Two separate possible barcode sequence classes were used, to generate two edit-directing plasmid pools distinguishable by their barcodes. One pool had barcodes of the form NNNNNNNNSWWS, while the other had barcodes of the form NNNNNNNNWSSW, where S can be either a cytosine or guanine base, W can be either an adenine or thymine base, while N can be any of the four bases. 10 micrograms of each PCR product was digested with MluI-HF and SphI-HF and gel extracted with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany).
20 micrograms of the first-step cloning product was digested with MluI-HF and SphI-HF (New England Biolabs, Ipswich, MA), treated with Shrimp Alkaline Phosphatase, and purified with the QIAquick PCR purification kit. 2 micrograms of the purified product was ligated with 0.7 micrograms of PCR-amplified insert (described above) to give the final barcoded edit-directing plasmid pool. Ligations were done in 800 microliter volumes with 8 microliters of T4 DNA ligase, at room temperature for 15 minutes. E. coli was then transformed with the ligation product. E. coli cells were grown in LB medium containing both 100 μg/mL Ampicillin and 50 μg/mL Kanamycin (Thermo Fisher Scientific, Waltham, MA). Plasmid DNA was extracted as above. We estimated that approximately 1.5E6 E. coli cells were transformed with each barcoded pool."
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