Cloning of sgRNAs into the LentiCRISPRv2 vector was performed as described (http://www.genome-engineering.org/crispr/ ). Briefly, the LentiCRISPRv2 plasmid was digested with BsmBI and gel‐purified. DNA oligonucleotides (Invitrogen) were annealed and ligated into the digested vector. Target sgRNA oligonucleotide sequences are listed in Figure S8 .
Dna oligonucleotides
Manufactured by Thermo Fisher Scientific
Sourced in China
DNA oligonucleotides are synthetic DNA sequences that are typically used as probes, primers, or for other research purposes in molecular biology. They are short, single-stranded DNA molecules that can be designed to target specific sequences within a larger DNA molecule.
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Lab products found in correlation
Primerquest tool, by Integrated DNA Technologies (2 mentions)
Idte buffer, by Integrated DNA Technologies (2 mentions)
Rna oligonucleotides, by Integrated DNA Technologies (2 mentions)
Oligoanalyzer tool, by Integrated DNA Technologies (2 mentions)
Topo ta cloning vector, by Thermo Fisher Scientific (2 mentions)
Nebuilder tool, by New England Biolabs (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Psilencer4.1 cmv vector, by Thermo Fisher Scientific (1 mentions)
Px330 u6 chimeric bb cbh hspcas9, by Addgene (1 mentions)
Dna oligonucleotide, by Integrated DNA Technologies (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Analytical grade inorganic chemicals, by Thermo Fisher Scientific (1 mentions)
Block it, by Thermo Fisher Scientific (1 mentions)
Pshuttle ires hrgfp 2 vector, by Agilent Technologies (1 mentions)
Padeasy vector, by Agilent Technologies (1 mentions)
19 protocols using dna oligonucleotides
1
CRISPR sgRNA Cloning into LentiCRISPRv2
2
DNA Oligonucleotide Synthesis Protocol
3
Synthetic miRNA spike-in screening
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Sequences of mature microRNAs were obtained from the miRBase release 22 (www.mirbase.org ). RNA oligonucleotides with 5′-phosphate were synthesized and quantified by Integrated DNA Technologies. Spike-in miRNA sequences were screened in silico for homology against human, mouse and rat miRBase records (Release 22) with the following parameters - search sequences: mature miRNAs, search method: SSEARCH, e-value cut-off: 100, max. no. of hits: 100. No significant homology was found. DNA oligonucleotides were synthesized and quantified by Invitrogen. Sequences are available in Supplementary file.
4
Oligonucleotide Design for miRNA Analyses
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RNA oligonucleotides of unedited miR-379, edited miR-379, miR-380, unedited miR-411, edited miR-411, and miR-758 were purchased from Integrated DNA Technologies, dissolved in IDTE buffer, pH 7.5 (Integrated DNA Technologies), and then diluted in 10-fold dilution series for RT-qPCR. For experiments with yeast RNA background, 100 ng total yeast RNA (#AM7118, Invitrogen, Thermo Scientific) was added during RT sample preparation. DNA oligonucleotides were purchased from Invitrogen. Two-tailed RT primers were designed based on a hairpin sequence published by Androvic et al. (2017) (link) with hemiprobes designed to bind unedited or edited miR-379, and primer arms optimized to prevent the formation of unwanted secondary structures. Secondary structures of RT primers as well as secondary structures and dimers for qPCR primers were calculated using the OligoAnalyzer tool (Integrated DNA Technologies). ZEN/Iowa Black FQ double-quenched FAM-coupled miR-379 hydrolysis probe was designed using the PrimerQuest tool (Integrated DNA Technologies) and purchased from Integrated DNA Technologies. Primers for pri-miR-379 RT-PCR were based on those published by Kawahara et al. (2008) (link), and primers for cloning were designed using the NEBuilder tool (New England Biolabs). All RNA and DNA oligonucleotide sequences are listed in Supplemental Tables 2 and 3 .
5
CRISPR Plasmid Construction and Validation
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All DNA oligonucleotides were synthesized by Invitrogen (Beijing, China). pCas9, psgRNA and pTarget plasmid were provided by Biomics Biotechnologies (China, Nantong).
Single-nucleotide mutated target fragment was cloned into linearized pTarget plasmid. The resulting fusion pTarget plasmid was sequenced for verification.
Single-nucleotide mutated target fragment was cloned into linearized pTarget plasmid. The resulting fusion pTarget plasmid was sequenced for verification.
6
Plasmid Vectors and Cell Lines
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The psiCheck-2 vector was purchased from Promega. The pSilencer4.1-CMV vector and pcDNA3.1 vector were obtained from Ambion. DNA oligonucleotides were obtained from Invitrogen. The HPV 16-positive SiHa cervical cancer cell line and HPV-negative C33A cervical cancer cell line were used in this study.
7
Genetic Characterization of Melanoma Cell Lines
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All primer sequences are listed in Supplementary table 2 .
For identification of BRAF amplification in A375BMR cell, genomic DNA
was isolated and quantitative PCR was performed with primers amplifying BRAF,
CRAF and LINE. For identification of the MEK1 mutation in 451LuBRcells, genomic DNA was isolated and MEK1 exon 2 was cloned into the TOPO
TA-cloning vector (450071, Invitrogen), which was followed by Sanger sequencing.
The presence of BRAFV600E/DK in Mel888BMR cell was
identified as previously described9 (link).
Cloning of sgRNAs into LentiCRISPRv2 vector was performed as described
(http://www.genome-engineering.org/crispr/ ). Briefly, the
LentiCRISPRv2 plasmid was digested with BsmBI and gel-purified.
DNA oligonucleotides (Invitrogen) were annealed and ligated into the digested
vector. Target sgRNA oligonucleotide sequences are listed inSupplementary table 2 .
The plasmid encoding the MITF-M isoform was previously described16 . Production of lentivirus was performed
as described previously28 (link).
For identification of BRAF amplification in A375BMR cell, genomic DNA
was isolated and quantitative PCR was performed with primers amplifying BRAF,
CRAF and LINE. For identification of the MEK1 mutation in 451LuBRcells, genomic DNA was isolated and MEK1 exon 2 was cloned into the TOPO
TA-cloning vector (450071, Invitrogen), which was followed by Sanger sequencing.
The presence of BRAFV600E/DK in Mel888BMR cell was
identified as previously described9 (link).
Cloning of sgRNAs into LentiCRISPRv2 vector was performed as described
(
LentiCRISPRv2 plasmid was digested with BsmBI and gel-purified.
DNA oligonucleotides (Invitrogen) were annealed and ligated into the digested
vector. Target sgRNA oligonucleotide sequences are listed in
The plasmid encoding the MITF-M isoform was previously described16 . Production of lentivirus was performed
as described previously28 (link).
8
Genetic Characterization of Melanoma Cell Lines
9
CRISPR/Cas9-Mediated Knockout of ITGB5 in Pigs
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Single-guide RNAs (sgRNAs) targeted to exon 1 and 2 of Sus scrofa integrin subunit beta 5 (ITGB5) were designed using online CRISPR design tools (http://crispr.mit.edu/ ) (50 (link)). Six sgRNAs (Figure 4A , Table 2 ) were selected for expression vector construction using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR–associated protein 9 (Cas9)–sgRNA, based on their predicted scores and lower off-target effects. DNA oligonucleotides corresponding to the sgRNAs were synthesized by Invitrogen (Shanghai, China). Annealed oligonucleotides were inserted into pX330-U6-Chimeric_BB-CBh-hSpCas9 (plasmid 42230, PX330, Addgene, a gift from Feng Zhang, Broad Institute of MIT and Harvard) containing two BbsI (R3539S, NEB, Ipswich, MA) restriction enzyme sites, using a published protocol (51 (link)). The sgRNA with higher efficiency was used for single clone selection.
10
Electrophoretic Mobility Shift Assay for Protein-DNA Interactions
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DNA oligonucleotides (Invitrogen) were annealed to form duplexes and end-labeled by T4 polynucleotide kinase (NEB) using γ33P ATP. The proteins were incubated with the nucleic acid probe for 15 minutes on ice in EMSA buffer [19 ] in the presence of 500 ng poly dI-dC. Either wild-type or mutant non-labeled competitor was added at a 50 times excess to two of the reactions while a third reaction was incubated with anti-ilf3 antibody to allow identification of the specific DNA-protein complex. After incubation the DNA and DNA-protein complexes were separated on a 4% native polyacrylamide gel in 0.25 X TBE. The gels were dried and visualized using a phosphorimager (Fuji).
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