Saline:Article Title: MapR: A Method for Identifying Native R-Loops Genome Wide.
Article Snippet: .. E. coli BL21 (DE3; Thermo Fisher, # C601003) pGEX-6P-1 GST-MNase (Addgene, # 136291) pGEX-6P-1 GST-RH -MNase (Addgene, # 136292) LB-ampicillin agar plates (100 μg/ml ampicillin), 37°C LB medium containing 100 μg/ml ampicillin 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Fisher Scientific, # BP1755) 1× phosphate-buffered saline (PBS; see recipe), 4°C Glutathione S-transferase (GST)-agarose beads (PrepEase Protein Purification Glutathione Agarose 4B, Affymetrix, # 78820) GST elution buffer (see recipe), 4°C 10% SDS-PAGE gel Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, # 5000006) BC100 (see recipe), 4°C Glycerol Binding buffer (see recipe) Concanavalin A–coated magnetic beads (BioMag Plus Concanavalin A, Polysciences, # 86057) Wash buffer (see recipe) Cells (any mammalian cell type) 0.05% (w/v) trypsin-EDTA Cell culture medium containing 10% (v/v) serum Dig-wash buffer (see recipe; make fresh) 0.1 M calcium chloride (CaCl2) Yan and Sarma 2 of 12 Current Protocols in Molecular Biology Figure 1 Schematic of MapR workflow and a brief experimental outline. .. 2× stop buffer (see recipe; make fresh) 10% (w/v) sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K Phenol/chloroform/isoamyl alcohol mix (25:24:1, v/v; Invitrogen, # 15593049) 7.5 M ammonium acetate (NH4OAc) Linear acrylamide (VWR, # 97063-560) 75% (v/v) and 100% ethanol, 4°C TE buffer Yan and Sarma 3 of 12 Current Protocols in Molecular Biology NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, # E7645S) NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, NEB, # E7600S) 37°C incubator 50-ml conical tube 37°C shaker Erlenmeyer flask Spectrophotometer Room-temperature or 4°C centrifuge Probe sonicator 1.5-ml Eppendorf tubes Room-temperature or 4°C microcentrifuge Rotator 10% SDS-PAGE gel Magnetic stand Light microscope Hemocytometer 37°C and 70°C heat blocks Phase Lock Gel Heavy tubes (Quantabio, # 2302830) TapeStation (Model 4200, Agilent) Additional reagents and equipment for SDS-PAGE and Coomassie staining (see Current Protocols article; Gallagher, 2012), Bradford assay (see Current Protocols article; Olson & Markwell, 2007), and dialysis (see Current Protocols article; Zumstein, 2001)
Protein Purification:Article Title: MapR: A Method for Identifying Native R-Loops Genome Wide.
Article Snippet: .. E. coli BL21 (DE3; Thermo Fisher, # C601003) pGEX-6P-1 GST-MNase (Addgene, # 136291) pGEX-6P-1 GST-RH -MNase (Addgene, # 136292) LB-ampicillin agar plates (100 μg/ml ampicillin), 37°C LB medium containing 100 μg/ml ampicillin 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Fisher Scientific, # BP1755) 1× phosphate-buffered saline (PBS; see recipe), 4°C Glutathione S-transferase (GST)-agarose beads (PrepEase Protein Purification Glutathione Agarose 4B, Affymetrix, # 78820) GST elution buffer (see recipe), 4°C 10% SDS-PAGE gel Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, # 5000006) BC100 (see recipe), 4°C Glycerol Binding buffer (see recipe) Concanavalin A–coated magnetic beads (BioMag Plus Concanavalin A, Polysciences, # 86057) Wash buffer (see recipe) Cells (any mammalian cell type) 0.05% (w/v) trypsin-EDTA Cell culture medium containing 10% (v/v) serum Dig-wash buffer (see recipe; make fresh) 0.1 M calcium chloride (CaCl2) Yan and Sarma 2 of 12 Current Protocols in Molecular Biology Figure 1 Schematic of MapR workflow and a brief experimental outline. .. 2× stop buffer (see recipe; make fresh) 10% (w/v) sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K Phenol/chloroform/isoamyl alcohol mix (25:24:1, v/v; Invitrogen, # 15593049) 7.5 M ammonium acetate (NH4OAc) Linear acrylamide (VWR, # 97063-560) 75% (v/v) and 100% ethanol, 4°C TE buffer Yan and Sarma 3 of 12 Current Protocols in Molecular Biology NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, # E7645S) NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, NEB, # E7600S) 37°C incubator 50-ml conical tube 37°C shaker Erlenmeyer flask Spectrophotometer Room-temperature or 4°C centrifuge Probe sonicator 1.5-ml Eppendorf tubes Room-temperature or 4°C microcentrifuge Rotator 10% SDS-PAGE gel Magnetic stand Light microscope Hemocytometer 37°C and 70°C heat blocks Phase Lock Gel Heavy tubes (Quantabio, # 2302830) TapeStation (Model 4200, Agilent) Additional reagents and equipment for SDS-PAGE and Coomassie staining (see Current Protocols article; Gallagher, 2012), Bradford assay (see Current Protocols article; Olson & Markwell, 2007), and dialysis (see Current Protocols article; Zumstein, 2001)
SDS Page:Article Title: MapR: A Method for Identifying Native R-Loops Genome Wide.
Article Snippet: .. E. coli BL21 (DE3; Thermo Fisher, # C601003) pGEX-6P-1 GST-MNase (Addgene, # 136291) pGEX-6P-1 GST-RH -MNase (Addgene, # 136292) LB-ampicillin agar plates (100 μg/ml ampicillin), 37°C LB medium containing 100 μg/ml ampicillin 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Fisher Scientific, # BP1755) 1× phosphate-buffered saline (PBS; see recipe), 4°C Glutathione S-transferase (GST)-agarose beads (PrepEase Protein Purification Glutathione Agarose 4B, Affymetrix, # 78820) GST elution buffer (see recipe), 4°C 10% SDS-PAGE gel Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, # 5000006) BC100 (see recipe), 4°C Glycerol Binding buffer (see recipe) Concanavalin A–coated magnetic beads (BioMag Plus Concanavalin A, Polysciences, # 86057) Wash buffer (see recipe) Cells (any mammalian cell type) 0.05% (w/v) trypsin-EDTA Cell culture medium containing 10% (v/v) serum Dig-wash buffer (see recipe; make fresh) 0.1 M calcium chloride (CaCl2) Yan and Sarma 2 of 12 Current Protocols in Molecular Biology Figure 1 Schematic of MapR workflow and a brief experimental outline. .. 2× stop buffer (see recipe; make fresh) 10% (w/v) sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K Phenol/chloroform/isoamyl alcohol mix (25:24:1, v/v; Invitrogen, # 15593049) 7.5 M ammonium acetate (NH4OAc) Linear acrylamide (VWR, # 97063-560) 75% (v/v) and 100% ethanol, 4°C TE buffer Yan and Sarma 3 of 12 Current Protocols in Molecular Biology NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, # E7645S) NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, NEB, # E7600S) 37°C incubator 50-ml conical tube 37°C shaker Erlenmeyer flask Spectrophotometer Room-temperature or 4°C centrifuge Probe sonicator 1.5-ml Eppendorf tubes Room-temperature or 4°C microcentrifuge Rotator 10% SDS-PAGE gel Magnetic stand Light microscope Hemocytometer 37°C and 70°C heat blocks Phase Lock Gel Heavy tubes (Quantabio, # 2302830) TapeStation (Model 4200, Agilent) Additional reagents and equipment for SDS-PAGE and Coomassie staining (see Current Protocols article; Gallagher, 2012), Bradford assay (see Current Protocols article; Olson & Markwell, 2007), and dialysis (see Current Protocols article; Zumstein, 2001)
Binding Assay:Article Title: MapR: A Method for Identifying Native R-Loops Genome Wide.
Article Snippet: .. E. coli BL21 (DE3; Thermo Fisher, # C601003) pGEX-6P-1 GST-MNase (Addgene, # 136291) pGEX-6P-1 GST-RH -MNase (Addgene, # 136292) LB-ampicillin agar plates (100 μg/ml ampicillin), 37°C LB medium containing 100 μg/ml ampicillin 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Fisher Scientific, # BP1755) 1× phosphate-buffered saline (PBS; see recipe), 4°C Glutathione S-transferase (GST)-agarose beads (PrepEase Protein Purification Glutathione Agarose 4B, Affymetrix, # 78820) GST elution buffer (see recipe), 4°C 10% SDS-PAGE gel Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, # 5000006) BC100 (see recipe), 4°C Glycerol Binding buffer (see recipe) Concanavalin A–coated magnetic beads (BioMag Plus Concanavalin A, Polysciences, # 86057) Wash buffer (see recipe) Cells (any mammalian cell type) 0.05% (w/v) trypsin-EDTA Cell culture medium containing 10% (v/v) serum Dig-wash buffer (see recipe; make fresh) 0.1 M calcium chloride (CaCl2) Yan and Sarma 2 of 12 Current Protocols in Molecular Biology Figure 1 Schematic of MapR workflow and a brief experimental outline. .. 2× stop buffer (see recipe; make fresh) 10% (w/v) sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K Phenol/chloroform/isoamyl alcohol mix (25:24:1, v/v; Invitrogen, # 15593049) 7.5 M ammonium acetate (NH4OAc) Linear acrylamide (VWR, # 97063-560) 75% (v/v) and 100% ethanol, 4°C TE buffer Yan and Sarma 3 of 12 Current Protocols in Molecular Biology NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, # E7645S) NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, NEB, # E7600S) 37°C incubator 50-ml conical tube 37°C shaker Erlenmeyer flask Spectrophotometer Room-temperature or 4°C centrifuge Probe sonicator 1.5-ml Eppendorf tubes Room-temperature or 4°C microcentrifuge Rotator 10% SDS-PAGE gel Magnetic stand Light microscope Hemocytometer 37°C and 70°C heat blocks Phase Lock Gel Heavy tubes (Quantabio, # 2302830) TapeStation (Model 4200, Agilent) Additional reagents and equipment for SDS-PAGE and Coomassie staining (see Current Protocols article; Gallagher, 2012), Bradford assay (see Current Protocols article; Olson & Markwell, 2007), and dialysis (see Current Protocols article; Zumstein, 2001)
Magnetic Beads:Article Title: MapR: A Method for Identifying Native R-Loops Genome Wide.
Article Snippet: .. E. coli BL21 (DE3; Thermo Fisher, # C601003) pGEX-6P-1 GST-MNase (Addgene, # 136291) pGEX-6P-1 GST-RH -MNase (Addgene, # 136292) LB-ampicillin agar plates (100 μg/ml ampicillin), 37°C LB medium containing 100 μg/ml ampicillin 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Fisher Scientific, # BP1755) 1× phosphate-buffered saline (PBS; see recipe), 4°C Glutathione S-transferase (GST)-agarose beads (PrepEase Protein Purification Glutathione Agarose 4B, Affymetrix, # 78820) GST elution buffer (see recipe), 4°C 10% SDS-PAGE gel Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, # 5000006) BC100 (see recipe), 4°C Glycerol Binding buffer (see recipe) Concanavalin A–coated magnetic beads (BioMag Plus Concanavalin A, Polysciences, # 86057) Wash buffer (see recipe) Cells (any mammalian cell type) 0.05% (w/v) trypsin-EDTA Cell culture medium containing 10% (v/v) serum Dig-wash buffer (see recipe; make fresh) 0.1 M calcium chloride (CaCl2) Yan and Sarma 2 of 12 Current Protocols in Molecular Biology Figure 1 Schematic of MapR workflow and a brief experimental outline. .. 2× stop buffer (see recipe; make fresh) 10% (w/v) sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K Phenol/chloroform/isoamyl alcohol mix (25:24:1, v/v; Invitrogen, # 15593049) 7.5 M ammonium acetate (NH4OAc) Linear acrylamide (VWR, # 97063-560) 75% (v/v) and 100% ethanol, 4°C TE buffer Yan and Sarma 3 of 12 Current Protocols in Molecular Biology NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, # E7645S) NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, NEB, # E7600S) 37°C incubator 50-ml conical tube 37°C shaker Erlenmeyer flask Spectrophotometer Room-temperature or 4°C centrifuge Probe sonicator 1.5-ml Eppendorf tubes Room-temperature or 4°C microcentrifuge Rotator 10% SDS-PAGE gel Magnetic stand Light microscope Hemocytometer 37°C and 70°C heat blocks Phase Lock Gel Heavy tubes (Quantabio, # 2302830) TapeStation (Model 4200, Agilent) Additional reagents and equipment for SDS-PAGE and Coomassie staining (see Current Protocols article; Gallagher, 2012), Bradford assay (see Current Protocols article; Olson & Markwell, 2007), and dialysis (see Current Protocols article; Zumstein, 2001)
Cell Culture:Article Title: MapR: A Method for Identifying Native R-Loops Genome Wide.
Article Snippet: .. E. coli BL21 (DE3; Thermo Fisher, # C601003) pGEX-6P-1 GST-MNase (Addgene, # 136291) pGEX-6P-1 GST-RH -MNase (Addgene, # 136292) LB-ampicillin agar plates (100 μg/ml ampicillin), 37°C LB medium containing 100 μg/ml ampicillin 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Fisher Scientific, # BP1755) 1× phosphate-buffered saline (PBS; see recipe), 4°C Glutathione S-transferase (GST)-agarose beads (PrepEase Protein Purification Glutathione Agarose 4B, Affymetrix, # 78820) GST elution buffer (see recipe), 4°C 10% SDS-PAGE gel Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, # 5000006) BC100 (see recipe), 4°C Glycerol Binding buffer (see recipe) Concanavalin A–coated magnetic beads (BioMag Plus Concanavalin A, Polysciences, # 86057) Wash buffer (see recipe) Cells (any mammalian cell type) 0.05% (w/v) trypsin-EDTA Cell culture medium containing 10% (v/v) serum Dig-wash buffer (see recipe; make fresh) 0.1 M calcium chloride (CaCl2) Yan and Sarma 2 of 12 Current Protocols in Molecular Biology Figure 1 Schematic of MapR workflow and a brief experimental outline. .. 2× stop buffer (see recipe; make fresh) 10% (w/v) sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K Phenol/chloroform/isoamyl alcohol mix (25:24:1, v/v; Invitrogen, # 15593049) 7.5 M ammonium acetate (NH4OAc) Linear acrylamide (VWR, # 97063-560) 75% (v/v) and 100% ethanol, 4°C TE buffer Yan and Sarma 3 of 12 Current Protocols in Molecular Biology NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, # E7645S) NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, NEB, # E7600S) 37°C incubator 50-ml conical tube 37°C shaker Erlenmeyer flask Spectrophotometer Room-temperature or 4°C centrifuge Probe sonicator 1.5-ml Eppendorf tubes Room-temperature or 4°C microcentrifuge Rotator 10% SDS-PAGE gel Magnetic stand Light microscope Hemocytometer 37°C and 70°C heat blocks Phase Lock Gel Heavy tubes (Quantabio, # 2302830) TapeStation (Model 4200, Agilent) Additional reagents and equipment for SDS-PAGE and Coomassie staining (see Current Protocols article; Gallagher, 2012), Bradford assay (see Current Protocols article; Olson & Markwell, 2007), and dialysis (see Current Protocols article; Zumstein, 2001)
Transformation Assay:Article Title: Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration.
Article Snippet: .. Briefly, E. coli BL21 transformed with pGEX-6P-1 GST-RH-MNase (Addgene, # 136292) were grown overnight, and protein expression was induced by adding 1 mM IPTG for 3 h. Bacterial cells were lysed by sonication, and the cleared lysate was incubated with glutathione sepharose beads overnight at 4 °C. .. Genomic DNA was resuspended in TE and spotted onto nylon membranes (Amersham Hybond-N, GE Healthcare, Cat# RPN303N).
Expressing:Article Title: Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration.
Article Snippet: .. Briefly, E. coli BL21 transformed with pGEX-6P-1 GST-RH-MNase (Addgene, # 136292) were grown overnight, and protein expression was induced by adding 1 mM IPTG for 3 h. Bacterial cells were lysed by sonication, and the cleared lysate was incubated with glutathione sepharose beads overnight at 4 °C. .. Genomic DNA was resuspended in TE and spotted onto nylon membranes (Amersham Hybond-N, GE Healthcare, Cat# RPN303N).
Sonication:Article Title: Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration.
Article Snippet: .. Briefly, E. coli BL21 transformed with pGEX-6P-1 GST-RH-MNase (Addgene, # 136292) were grown overnight, and protein expression was induced by adding 1 mM IPTG for 3 h. Bacterial cells were lysed by sonication, and the cleared lysate was incubated with glutathione sepharose beads overnight at 4 °C. .. Genomic DNA was resuspended in TE and spotted onto nylon membranes (Amersham Hybond-N, GE Healthcare, Cat# RPN303N).
Incubation:Article Title: Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration.
Article Snippet: .. Briefly, E. coli BL21 transformed with pGEX-6P-1 GST-RH-MNase (Addgene, # 136292) were grown overnight, and protein expression was induced by adding 1 mM IPTG for 3 h. Bacterial cells were lysed by sonication, and the cleared lysate was incubated with glutathione sepharose beads overnight at 4 °C. .. Genomic DNA was resuspended in TE and spotted onto nylon membranes (Amersham Hybond-N, GE Healthcare, Cat# RPN303N).
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