Journal: Cell reports methods

Article Title: Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

doi: 10.1016/j.crmeth.2023.100628

Figure Lengend Snippet: Figure 2. Functional score (FS) distribution and the correlation of FS between three assays (A) The distribution of FSs calculated from HAT, cisplatin, and olaparib sensitivity of mESCs with different BRCA2 variants. The color codes demarcate controls and variants of uncertain significance (VUSs). Variants that were classified as benign or likely benign in ClinVar were used as neutral/functional controls, and those that were classified as pathogenic or likely pathogenic were used as pathogenic/non-functional controls. A total of 223 variants were analyzed. (B) Correlation of FSs from three different assays (sensitivity to HAT, cisplatin, or olaparib) are plotted to compare the results of different assays. The Spearman correlation coefficient is displayed in each plot.

Article Snippet: For selecting the BRCA2 expressing clones, protein isolation and Western blot was carried out as described before.9 Rabbit polyclonal BRCA2 (recognizes an epitope between residues 450–500) antibody (BETHYL lab, Cat # A303-434A-T-1, 1:2000 dilution), and mouse monoclonal Vinculin antibody Cell Reports Methods 3, 100628, November 20, 2023 e3 (Santa Cruz biotech, Cat# sc25336, 1:200,000 dilution) were used to detect proteins.

Techniques: Functional Assay

Journal: Cell reports methods

Article Title: Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

doi: 10.1016/j.crmeth.2023.100628

Figure Lengend Snippet: Figure 5. Position of classified variants in BRCA2 (A) BRCA2 domains are marked with colored rectangles, with the amino acid (aa) position marked in parentheses. The variants located in different regions are marked below. OB, oligonucleotide/oligosaccharides binding; CTRB, C-terminal RAD51 binding. (B) Position of the splicing variants analyzed in this study. Functional variants are marked in green, non-functionals in magenta, and indeterminants in saffron.

Article Snippet: For selecting the BRCA2 expressing clones, protein isolation and Western blot was carried out as described before.9 Rabbit polyclonal BRCA2 (recognizes an epitope between residues 450–500) antibody (BETHYL lab, Cat # A303-434A-T-1, 1:2000 dilution), and mouse monoclonal Vinculin antibody Cell Reports Methods 3, 100628, November 20, 2023 e3 (Santa Cruz biotech, Cat# sc25336, 1:200,000 dilution) were used to detect proteins.

Techniques: Binding Assay, Functional Assay

Journal: Cell reports methods

Article Title: Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

doi: 10.1016/j.crmeth.2023.100628

Figure Lengend Snippet: Figure 2. Functional score (FS) distribution and the correlation of FS between three assays (A) The distribution of FSs calculated from HAT, cisplatin, and olaparib sensitivity of mESCs with different BRCA2 variants. The color codes demarcate controls and variants of uncertain significance (VUSs). Variants that were classified as benign or likely benign in ClinVar were used as neutral/functional controls, and those that were classified as pathogenic or likely pathogenic were used as pathogenic/non-functional controls. A total of 223 variants were analyzed. (B) Correlation of FSs from three different assays (sensitivity to HAT, cisplatin, or olaparib) are plotted to compare the results of different assays. The Spearman correlation coefficient is displayed in each plot.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains One shot Top10 competent cells Invitrogen Cat# C404003 E. coli SW102 National Cancer Institute NCI recombineering website: https://redrecombineering.ncifcrf.gov/ Chemicals, peptides, and recombinant proteins Gelatin (0.1%) Stemcell Technologies Cat# 07903 Knockout Dulbecco’s Modified Eagle Medium (DMEM) Gibco Cat# 10829-018 FBS (Hi Performance) Gibco Cat# 16000-044 b-mercaptoethanol Sigma Cat# M3148 Glutamine Penicillin Streptomycin Thermo Fisher Scientific Cat# 10378016 Trypsin EDTA (0.5%), no phenol red Thermo Fisher Scientific Cat# 15400054 DPBS (1X) Gibco Cat# 14190-144 Dimethyl sulfoxide (DMSO) Millipore Sigma Cat# D2650 Cisplatin Sigma Aldrich Cat# P4394 Olaparib Selleckchem Cat# AZD2281 G418 Thermo Fisher Scientific Cat# 10131035 Ampicillin Sigma Aldrich Cat# A9393 Galactose Sigma Aldrich Cat# 48260 2-deoxy-galactose Sigma Aldrich Cat# D4407 Biotin Sigma Aldrich Cat# B4501 L-Leucine Sigma Aldrich Cat# L8000 Kanamycin Sigma Aldrich Cat# K1637 Glycerol VWR Cat# J61059.K2 (NH4)2SO4 VWR Cat# BDH9216 KH2PO4 VWR Cat# BDH9268 FeSO4,7H2O VWR Cat# 95033-256 MgSO4$7H2O VWR Cat# MK569124 KOH VWR Cat# BDH9262 Bacto-tryptone VWR Cat# 76628 Yeast extract VWR Cat# AAJ60287-36 NaCl VWR Cat# BDH9286 Water Thermo Fisher Scientific Cat# 10977015 KH2PO4 VWR Cat# BDH9268 Na2HPO4,7H2O VWR Cat# 95035-872 NH4Cl VWR Cat# BDH9208 Agarose VWR Cat# 97064 Antibodies Rabbit polyclonal BRCA2 Bethyl Lab Cat # A303-434A-T-1, RRID: AB_3073617 Mouse monoclonal Vinculin antibody Santa Cruz Biotech Cat# sc25336, RRID: AB_628438 Goat anti-rabbit HRP conjugated Thermo Fisher Scientific Cat# 31460, RRID:AB_228341 Goat anti-mouse HRP conjugated Thermo Fisher Scientific Cat# 31430, RRID: AB_228307 Recombinant DNA pGalK National Cancer Institute NCI recombineering website: https://redrecombineering.ncifcrf.gov/ (Continued on next page) Cell Reports Methods 3, 100628, November 20, 2023 e1

Techniques: Functional Assay

Journal: Cell reports methods

Article Title: Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

doi: 10.1016/j.crmeth.2023.100628

Figure Lengend Snippet: Figure 5. Position of classified variants in BRCA2 (A) BRCA2 domains are marked with colored rectangles, with the amino acid (aa) position marked in parentheses. The variants located in different regions are marked below. OB, oligonucleotide/oligosaccharides binding; CTRB, C-terminal RAD51 binding. (B) Position of the splicing variants analyzed in this study. Functional variants are marked in green, non-functionals in magenta, and indeterminants in saffron.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains One shot Top10 competent cells Invitrogen Cat# C404003 E. coli SW102 National Cancer Institute NCI recombineering website: https://redrecombineering.ncifcrf.gov/ Chemicals, peptides, and recombinant proteins Gelatin (0.1%) Stemcell Technologies Cat# 07903 Knockout Dulbecco’s Modified Eagle Medium (DMEM) Gibco Cat# 10829-018 FBS (Hi Performance) Gibco Cat# 16000-044 b-mercaptoethanol Sigma Cat# M3148 Glutamine Penicillin Streptomycin Thermo Fisher Scientific Cat# 10378016 Trypsin EDTA (0.5%), no phenol red Thermo Fisher Scientific Cat# 15400054 DPBS (1X) Gibco Cat# 14190-144 Dimethyl sulfoxide (DMSO) Millipore Sigma Cat# D2650 Cisplatin Sigma Aldrich Cat# P4394 Olaparib Selleckchem Cat# AZD2281 G418 Thermo Fisher Scientific Cat# 10131035 Ampicillin Sigma Aldrich Cat# A9393 Galactose Sigma Aldrich Cat# 48260 2-deoxy-galactose Sigma Aldrich Cat# D4407 Biotin Sigma Aldrich Cat# B4501 L-Leucine Sigma Aldrich Cat# L8000 Kanamycin Sigma Aldrich Cat# K1637 Glycerol VWR Cat# J61059.K2 (NH4)2SO4 VWR Cat# BDH9216 KH2PO4 VWR Cat# BDH9268 FeSO4,7H2O VWR Cat# 95033-256 MgSO4$7H2O VWR Cat# MK569124 KOH VWR Cat# BDH9262 Bacto-tryptone VWR Cat# 76628 Yeast extract VWR Cat# AAJ60287-36 NaCl VWR Cat# BDH9286 Water Thermo Fisher Scientific Cat# 10977015 KH2PO4 VWR Cat# BDH9268 Na2HPO4,7H2O VWR Cat# 95035-872 NH4Cl VWR Cat# BDH9208 Agarose VWR Cat# 97064 Antibodies Rabbit polyclonal BRCA2 Bethyl Lab Cat # A303-434A-T-1, RRID: AB_3073617 Mouse monoclonal Vinculin antibody Santa Cruz Biotech Cat# sc25336, RRID: AB_628438 Goat anti-rabbit HRP conjugated Thermo Fisher Scientific Cat# 31460, RRID:AB_228341 Goat anti-mouse HRP conjugated Thermo Fisher Scientific Cat# 31430, RRID: AB_228307 Recombinant DNA pGalK National Cancer Institute NCI recombineering website: https://redrecombineering.ncifcrf.gov/ (Continued on next page) Cell Reports Methods 3, 100628, November 20, 2023 e1

Techniques: Binding Assay, Functional Assay

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of full-length BRCA2-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.

Article Snippet: Antibody , Rabbit polyclonal Anti-BRCA2 , Abcam , ab27976 , WB: mouse BRCA2 (1:1000).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of full-length BRCA2-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.

Article Snippet: Antibody , Rabbit polyclonal Anti-BRCA2 , Abcam , ab27976 , WB: mouse BRCA2 (1:1000).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.

Article Snippet: Antibody , Rabbit polyclonal Anti-BRCA2 , Abcam , ab27976 , WB: mouse BRCA2 (1:1000).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.

Article Snippet: Antibody , Rabbit polyclonal Anti-BRCA2 , Abcam , ab27976 , WB: mouse BRCA2 (1:1000).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal Anti-BRCA2 , Abcam , ab27976 , WB: mouse BRCA2 (1:1000).

Techniques: Suspension, Recombinant, Plasmid Preparation, Expressing, Modification, Knock-In, Construct, Sequencing, Mutagenesis, Transfection, Software