H358 BRMS1v2A273V/A273V isogenic cells were generated using CRISPR knock-in. Briefly, H358 cells were grown at 60%-80% confluence in a 100-mm culture dish 1 day before electroporation. On the day of electroporation, the ribonucleoprotein complexes were assembled by mixing Alt-R Cas9 nuclease (200 pmol) and Alt-R sgRNA (200 pmol; Integrated DNA Technologies). Approximately 1 × 106 cells were electroporated on the Neon transfection system using the Neon transfection system 100 μL kit (Thermo Fisher). A single-stranded oligonucleotide donor containing 172 base pairs as the homology-directed repair template was synthesized by Integrated DNA Technologies and added into the electroporation at a concentration of 2 μM. At 2 days after electroporation, cells were diluted, and single-cell clones were generated. The sequences of sgRNA and homology-directed repair template are listed in Supplementary Table 5 . The presence of SNP rs1052566 was screened by TaqMan SNP genotyping assays and confirmed by Sanger sequencing.
Alt r sgrna
Manufactured by Integrated DNA Technologies
Sourced in United States
The Alt-R sgRNA is a laboratory reagent used for gene editing. It serves as a guide RNA component in the CRISPR-Cas9 system, directing the Cas9 enzyme to targeted DNA sequences for efficient genome modifications.
Automatically generated - may contain errors
Lab products found in correlation
Alt r crrna, by Integrated DNA Technologies (2 mentions)
Idt duplex buffer, by Integrated DNA Technologies (2 mentions)
Alt r cas12a crrnas, by Integrated DNA Technologies (2 mentions)
Alt r tracrrna, by Integrated DNA Technologies (2 mentions)
Neon transfection system 100 μl kit, by Thermo Fisher Scientific (1 mentions)
Alt r grna, by Integrated DNA Technologies (1 mentions)
3 protocols using alt r sgrna
1
CRISPR-Mediated Isogenic Cell Line Generation
2
CRISPR-Cas9/Cas12a Ribonucleoprotein Preparation
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Cas9 gRNAs were prepared by mixing equimolar amounts of Alt-R crRNA and Alt-R tracrRNA (Integrated DNA Technologies, Coralville, IA, USA) in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate; Integrated DNA Technologies), heating to 95°C and slowly cooling to room temperature or using Alt-R sgRNA (Integrated DNA Technologies) hydrated in IDTE (pH 7.5) (10 mM Tris, pH 7.5, 0.1 mM EDTA; Integrated DNA Technologies). Cas12a gRNAs consisted of Alt-R Cas12a crRNAs (Integrated DNA Technologies) hydrated in IDTE (pH 7.5). RNP complexes were assembled by combining the CRISPR-Cas nuclease (Alt-R S.p. Cas9 Nuclease V3 or Alt-R A.s. Cas12a Ultra V3; Integrated DNA Technologies) and the Alt-R gRNA at a 1.2:1 molar ratio of gRNA:protein and incubating at room temperature for 10 min. The target-specific sequences of the gRNAs used in this study are listed in Tables S1 for Cas9 and S2 for Cas12a. The guides chosen were either within the same general genetic context (same amplicon sequencing space; enzyme dependent) or identical between the two cell lines (cell-line dependent) used in this study.
3
Streamlined CRISPR-Cas Ribonucleoprotein Assembly
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Cas9 gRNAs were prepared by mixing equimolar amounts of Alt-R™ crRNA and Alt-R tracrRNA (Integrated DNA Technologies, Coralville, IA, USA) in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate; Integrated DNA Technologies), heating to 95 °C and slowly cooling to room temperature or using Alt-R sgRNA (Integrated DNA Technologies) hydrated in IDTE pH 7.5 (10 mM Tris, pH 7.5, 0.1 mM EDTA; Integrated DNA Technologies). Cas12a gRNAs consisted of Alt-R Cas12a crRNAs (Integrated DNA Technologies) hydrated in IDTE pH 7.5. RNP complexes were assembled by combining the CRISPR–Cas nuclease (Alt-R S.p. Cas9 Nuclease V3, Alt-R S.p. HiFi Cas9 Nuclease V3, Alt-R S.p. Cas9 D10A V3, Alt-R S.p. Cas9 H840A V3, Alt-R A.s. Cas12a V3, or Alt-R A.s. Cas12a Ultra; Integrated DNA Technologies) and the Alt-R gRNA at a 1:1 to 1.2:1 molar ratio of gRNA:protein and incubating at room temperature for 30 min. For paired nicking experiments, each RNP was formed separately, and two RNPs were mixed together at an equal molar ratio prior to adding to the cells at the time of transfection. The 20-nt target specific sequences of the gRNAs used in this study are listed in Supplementary Table 1 .
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