Journal: PLoS Genetics

Article Title: Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

doi: 10.1371/journal.pgen.1005310

Figure Lengend Snippet: Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.

Article Snippet: The insert was then digested by BsaI (New England Biolabs, Ipswich, MA), whereas the vector was digested by BsmBI (New England Biolabs).

Techniques: Mutagenesis, Generated, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Infection, Sequencing, Amplification, Multiplex Assay

Journal: Science Advances

Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

doi: 10.1126/sciadv.1600699

Figure Lengend Snippet: gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

Article Snippet: Bsm BI/Aat II digestion The PCR product was digested with 10 μl of Bsm BI (10 U/μl; NEB) in 1× NEBuffer 3.1 in a 100-μl volume at 55°C for 6 hours, and then 5 μl of Aat II (20 U/μl; NEB) was added to the solution, which was left at 37°C overnight.

Techniques: Sequencing, Polymerase Chain Reaction, Fractionation, Marker

Journal: Nucleic Acid Therapeutics

Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

doi: 10.1089/nat.2014.0514

Figure Lengend Snippet: Schematic diagram of the RNA interference expression vector pRNAi-U6H1/Neo. The vector contains a multiple cloning sites with two BsmB I restriction sites to permit cloning of Fok I-digested sRNA fragments and an Sfi I site to assist in screening for recombinants.

Article Snippet: Fok I-digested fragments were ligated into linearized pRNAi-U6H1/Neo. pRNAi-U6H1/Neo was digested with BsmB I and dephosphorylated with calf intestinal alkaline phosphatase according to manufacturer's (NEB) protocols.

Techniques: Expressing, Plasmid Preparation, Clone Assay