The peGFP-RAD51AP1 expression vector is based on peGFP-C1 (Clontech) and has been described previously (Modesti et al., 2007 (link)). RAD51AP1 single or RAD54L/RAD51AP1 double KO cells were transfected with peGFP-C1 or peGFP-RAD51AP1 and Lipofectamine2000 (Invitrogen). Twenty-four hours after transfection, cells were subjected to a medium change, treated with 0.5 µM MMC for 2 h or 10 µM olaparib for 24 h. Cells were washed twice with warm PBS, fresh medium was added, and cells were incubated for the times indicated. Cells were lysed in chilled lysis buffer containing 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 0.5% NP-40, supplemented with EDTA-free protease inhibitor cocktail (Roche) and HALT phosphatase inhibitors (Thermo Fisher Scientific). For 1.5 × 106 cells, 25 μl of GFP-Trap® dynabeads (ChromoTek) were used to trap the ectopic proteins. Protein lysates were diluted to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, and 0.1 unit DNase I (Gold Biotechnology) per µg protein, and mixed with the equilibrated beads at 4°C for 1 h with gentle rotation. The GFP-Trap® dynabeads were washed three times with 500 µl binding buffer, bound protein complexes were eluted in 40 µl 2× LDS buffer (Thermo Fisher Scientific) and fractionated on 7% NuPAGE Tris-Acetate gels (Thermo Fisher Scientific) and for Western blot analysis.
Dnase 1
Manufactured by GoldBio
Sourced in United States
DNase I is a deoxyribonuclease enzyme that catalyzes the hydrolytic cleavage of DNA. It is commonly used in molecular biology and biochemistry applications to degrade DNA.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Halt phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Lds buffer, by Thermo Fisher Scientific (2 mentions)
Pegfp c1, by Takara Bio (2 mentions)
Gfp trap dynabeads, by Proteintech (2 mentions)
Edta free protease inhibitor cocktail, by Roche (2 mentions)
Gfp trap dynabeads, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Pegfp c1, by Thermo Fisher Scientific (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
70 m cell strainer, by BD (1 mentions)
Rbc lysis buffer, by Miltenyi Biotec (1 mentions)
Dead cell removal kit, by Miltenyi Biotec (1 mentions)
Powertrack sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Tripure reagent, by Roche (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
E coli poly a polymerase, by New England Biolabs (1 mentions)
Protoscript 2 reverse transcriptase, by New England Biolabs (1 mentions)
Nupage tris acetate gel, by Thermo Fisher Scientific (1 mentions)
6 protocols using dnase 1
1
Purification and Analysis of GFP-RAD51AP1 Complexes
2
Isolation of Small Intestinal Crypts and Single Cells
3
Single-Cell Tumor Dissociation Protocol
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Single-cell preparation for scRNA-seq and mass cytometry validation was performed according to a previous study [16 (link)]. In brief, tumors were cut into approximately 1 mm3 pieces and digested in Hank’s balanced salt solution (HBSS) containing 1.5 mg/ml collagenase IV (Sigma‒Aldrich), 1 mg/ml hyaluronidase (Sigma‒Aldrich) and 500 µg/ml DNase I (GoldBio) while rotating at 200 rpm and 37 °C for 30 min. Cell suspensions were subsequently passed through a 70-µm cell strainer (BD, Biosciences), followed by centrifugation at 400 × g for 10 min. Red blood cells were lysed with RBC lysis buffer (Miltenyi Biotec, 130-094-183) for 2 min on ice, and dead cells were removed with a dead cell removal kit (Miltenyi Biotec, 130-090-101) following the manufacturer’s instructions.
4
Isolation of Small Intestinal Crypts and Single Cells
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Small intestinal crypts and single GFP positive cells were isolated as previously described 7 (link). The entire small intestine was harvested, opened longitudinally and washed with PBS. Mucus and villi were scraped and discarded using a thin glass coverslip and tissue was cut into small (approximately 2cm) sections. These were washed again in PBS and incubated in 2.5 mM EDTA diluted in PBS for 1 hour with gentle agitation at 4 degree C. Crypts were liberated and collected by centrifugation (400g for 5 minutes at 4 degree C) followed by washing with PBS to remove EDTA. Crypts in PBS were passed through a 70-um cell strainer and centrifuged as before. This pellet was resuspended in 50% culture media (described below) and 50% Corning Growth Factor Reduced matrigel (Fisher Scientific #356231) for culture or dissociated for single cells. Single cell isolation was carried out as described previously 8 (link) by resuspending crypts in TrypLE Express (Invitrogen #1260413) with DNase I (Gold Biotechnology #D-300–1) for 10 minutes at 37 degree C. Dissociated cells were washed in culture media without growth factors, centrifuged at 700g, resuspended and passed through a 35-um strainer (Fisher Scientific #08–771-23) and analyzed by FACS. Neagtive staining was carried out with DAPI and single live cells were collected, pelleted, and snap frozen for analysis or resuspended for culture.
5
Wheat RNA Extraction and RT-qPCR Analysis
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Total RNA was isolated from fully expanded upper leaves of wheat infected with WSMV, TriMV, WSMV plus TriMV, or mock-inoculated wheat plants using TRIpure reagent (Roche, IN, USA), followed by treatment with DNAse I (GoldBio, MO, USA). One microgram of the total RNA was polyadenylated using E. coli Poly(A) polymerase (New England Biolabs, MA, USA), and the first-strand cDNA was subsequently synthesized using ProtoScript II Reverse Transcriptase (New England Biolabs, MA, USA) and poly(T) adapter (Table S4 ).
For RT-qPCR, 1 ng template cDNA, miRNA-specific forward miRNA primer, and pTR (5 mM each) (Table S4 ) were mixed with PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific, MA, USA) in a final volume of 20 µl. Three biological replicates were employed for each treatment, with two technical replicates for each biological replicate. BioRad CFX96 was used with a standard-short protocol for the amplification of miRNA templates. Briefly, a single step of 2 min at 95 °C was used for enzyme activation, followed by 40 cycles of 15 s at 95 °C and 30 s at 55 °C, and a standard dissociation step (i.e., 65 °C to 95 °C, 0.5 °C increments). For each biological replicate, gene expression (ΔCt) was calculated by subtracting Ct values of the normalization gene (5.8S ribosomal RNA) from Ct values of inoculated (virus- or mock-) samples.
For RT-qPCR, 1 ng template cDNA, miRNA-specific forward miRNA primer, and pTR (5 mM each) (Table S
6
Purification and Analysis of RAD51AP1 Complex
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The peGFP-RAD51AP1 expression vector is based on peGFP-C1 (Clontech) and has been described previously (Modesti et al. 2007) . RAD54/RAD51AP1 double KO cells were transfected with peGFP-C1 or peGFP-RAD51AP1 and Lipofectamine2000 (Invitrogen). Twenty-four hours after transfection, cells were subjected to a medium change or treated with 0.5 µM MMC for 2 hours. Cells were washed twice with warm PBS, fresh medium was added, and cells were incubated for the times indicated. Cells were lysed in chilled lysis buffer containing 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 0.5% NP-40, supplemented with EDTA-free protease inhibitor cocktail (Roche) and HALT phosphatase inhibitors (Thermo Fisher Scientific). For 1.5x10 6 cells, 25 μl of GFP-Trap ® dynabeads (ChromoTek) were used to trap the ectopic proteins. Protein lysates were diluted to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, and 0.1unit DNase I (Gold Biotechnology) per µg protein, and mixed with the equilibrated beads at 4°C for 1 h with gentle rotation. The GFP-Trap ® dynabeads were washed three times with 500 µl binding buffer, bound protein complexes were eluted in 40 µl 2× LDS buffer (Thermo Fisher Scientific) and fractionated on 7% NuPAGE Tris-Acetate gels (Thermo Fisher Scientific) and for Western blot analysis.
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