pcr primers n a n a fluidigm access array barcodes fluidigm 101 0744 recombinant dna pspgrna addgene  (Addgene inc)

Journal: Molecular cell

Article Title: Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities

doi: 10.1016/j.molcel.2020.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-FLAG Sigma F1804 Anti-β-actin Cell Signaling 4970 Chemicals, Peptides, and Recombinant Proteins T4 DNA ligase NEB M0202 T7 DNA ligase NEB M0318 Phusion DNA polymerase NEB M0530 Gibson Assembly Mastermix NEB E2611 AgeI restriction endonuclease NEB R0552 NotI-HF restriction endonuclease NEB R0189 EcoRI-HR restriction endonuclease NEB R3101 BamHI restriction endonuclease NEB R3136 BpiI restriction endonuclease NEB ER1011 Calf intestinal phosphatase (CIP) NEB M0525 Ribonucleotide solution mix NEB N0466 T7 RNAP Dr. Miljan Simonovic N/A 0.1% Gelatin Millipore ES-006-B Knockout DMEM GIBCO 10829–018 L-Glutamine GIBCO 25030–081 HEPES Thermo Fisher Scientific SH30237.01 MEM NEAA GIBCO 11140 LIF Millipore ESG1106 2-mercaptoethanol GIBCO 21985–023 CHIR99021 Sigma SML1046 Trypsin-EDTA GIBCO 25200–072 Pen Strep GIBCO 15140 QuickExtract DNA Extraction Solution Epicenter BQ0901 PD0325901 Adooq A10256 N2 supplement (100x) Thermo Fisher Scientific 17502058 B27 supplement (50x) Thermo Fisher Scientific 17504001 Poly-l-lysine (0.01%) Millipore Sigma 25988–63-0 High glucose DMEM Gibco 11965118 Fetal bovine serum Gibco 26140079 Lipofectamine 2000 Thermo Fisher Scientific 11668019 Optimem Gibco 11058021 Hoeschst33342 Thermo Fisher Scientific H3570 Ouabain Tocris 1076 Calcein AM Thermo Fisher Scientific C1430 Accuprime II Thermo Fisher Scientific 12–341-012 AMPure XP beads Beckman Coulter A63880 Critical Commercial Assays Zymo RNA Clean & Concentrator Zymo Research R1013 Zymo DNA Clean & Concentrator Zymo Research D4031 Deposited Data Raw Data (unprocessed gels) Mendeley doi: 10.17632/tz4yxtwrcc.1 Amplicon sequencing data (pGuide genes and genomic target sequences) NCBI SAMN16122927 Experimental Models: Cell Lines Mouse embryonic stem cells (mESC) N/A N/A HEK293 Dr. Sojin Shikano N/A HEK293::TLR Dr. William Putzbach N/A 4T1::EGFP Dr. Nissim Hay N/A mESC harboring Rex1::EGFPd2 Dr. Austin Smith doi: 10.1038/ncb2267 Experimental Models: Organisms/Strains Oligonucleotides See Table S1 for pGuide sequences and Cdt1/Gem degrons N/A N/A See Table S2 for PCR primers N/A N/A Fluidigm Access Array Barcodes Fluidigm 101–0744 Recombinant DNA pSpgRNA Addgene #47018 doi: 10.1038/nmeth.2600 PB-U6insert-EF1puro (piggybac transposon vector) Addgene #104537 doi: 10.1126/science.aat9804 Super pigggybac transposase SBI PB210PA-1 pX330 Addgene #42230 doi: 10.1126/science.1231143 Software and Algorithms CytExpert Beckman Coulter CRISPresso version 2 N/A doi: 10.1038/s41587-019-0032-3 Other Open in a separate window KEY RESOURCES TABLE.

Techniques: Recombinant, Knock-Out, DNA Extraction, Amplification, Sequencing, Plasmid Preparation, Software

Journal: Molecular cell

Article Title: Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities

doi: 10.1016/j.molcel.2020.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-FLAG Sigma F1804 Anti-β-actin Cell Signaling 4970 Chemicals, Peptides, and Recombinant Proteins T4 DNA ligase NEB M0202 T7 DNA ligase NEB M0318 Phusion DNA polymerase NEB M0530 Gibson Assembly Mastermix NEB E2611 AgeI restriction endonuclease NEB R0552 NotI-HF restriction endonuclease NEB R0189 EcoRI-HR restriction endonuclease NEB R3101 BamHI restriction endonuclease NEB R3136 BpiI restriction endonuclease NEB ER1011 Calf intestinal phosphatase (CIP) NEB M0525 Ribonucleotide solution mix NEB N0466 T7 RNAP Dr. Miljan Simonovic N/A 0.1% Gelatin Millipore ES-006-B Knockout DMEM GIBCO 10829–018 L-Glutamine GIBCO 25030–081 HEPES Thermo Fisher Scientific SH30237.01 MEM NEAA GIBCO 11140 LIF Millipore ESG1106 2-mercaptoethanol GIBCO 21985–023 CHIR99021 Sigma SML1046 Trypsin-EDTA GIBCO 25200–072 Pen Strep GIBCO 15140 QuickExtract DNA Extraction Solution Epicenter BQ0901 PD0325901 Adooq A10256 N2 supplement (100x) Thermo Fisher Scientific 17502058 B27 supplement (50x) Thermo Fisher Scientific 17504001 Poly-l-lysine (0.01%) Millipore Sigma 25988–63-0 High glucose DMEM Gibco 11965118 Fetal bovine serum Gibco 26140079 Lipofectamine 2000 Thermo Fisher Scientific 11668019 Optimem Gibco 11058021 Hoeschst33342 Thermo Fisher Scientific H3570 Ouabain Tocris 1076 Calcein AM Thermo Fisher Scientific C1430 Accuprime II Thermo Fisher Scientific 12–341-012 AMPure XP beads Beckman Coulter A63880 Critical Commercial Assays Zymo RNA Clean & Concentrator Zymo Research R1013 Zymo DNA Clean & Concentrator Zymo Research D4031 Deposited Data Raw Data (unprocessed gels) Mendeley doi: 10.17632/tz4yxtwrcc.1 Amplicon sequencing data (pGuide genes and genomic target sequences) NCBI SAMN16122927 Experimental Models: Cell Lines Mouse embryonic stem cells (mESC) N/A N/A HEK293 Dr. Sojin Shikano N/A HEK293::TLR Dr. William Putzbach N/A 4T1::EGFP Dr. Nissim Hay N/A mESC harboring Rex1::EGFPd2 Dr. Austin Smith doi: 10.1038/ncb2267 Experimental Models: Organisms/Strains Oligonucleotides See Table S1 for pGuide sequences and Cdt1/Gem degrons N/A N/A See Table S2 for PCR primers N/A N/A Fluidigm Access Array Barcodes Fluidigm 101–0744 Recombinant DNA pSpgRNA Addgene #47018 doi: 10.1038/nmeth.2600 PB-U6insert-EF1puro (piggybac transposon vector) Addgene #104537 doi: 10.1126/science.aat9804 Super pigggybac transposase SBI PB210PA-1 pX330 Addgene #42230 doi: 10.1126/science.1231143 Software and Algorithms CytExpert Beckman Coulter CRISPresso version 2 N/A doi: 10.1038/s41587-019-0032-3 Other Open in a separate window KEY RESOURCES TABLE.

Techniques: Recombinant, Knock-Out, DNA Extraction, Amplification, Sequencing, Plasmid Preparation, Software