U2OS and Flp-in 293 cells were cultured in Dulbecco’s modified Eagle medium (catalog no. 12-604F, Lonza) with 10% (volume [vol]/vol) fetal bovine serum at 37 °C with 5% CO2. The U2OS–TRE cells used for the DART system have been described in previous articles (28 (link)). For plasmid and siRNA transfection, Lipofectamine 2000 and Lipofectamine RNAiMax (Invitrogen) were used following the manufacturer’s standard protocol, respectively. The siRNA for TRDMT1 was purchased from Invitrogen (siRNA ID s4219; catalog no. 4392420). The siRNA for FMRP was purchased from Dharmacon (catalog no. L-019631-00-0005); the sequences of siRNA targeting FMRP 3′ UTR are 5′- GUACUGAGCAGUGAUAUUCdTdT-3′ and 5′- GAAUAUCACUGCUCAGUACdTdT-3′. Other siRNAs include siTET1 (catalog no. AM16708, assay ID:147892, Thermo), siBRCA1 (catalog no. L-003461-00, Dharmacon), and siBRCA2 (catalog no. GS675, Qiagen). Antibodies used in this study are summarized in SI Appendix , Table 1. Other methods used in the study are described in detail in SI Appendix , Materials and Methods.
Sibrca2
Manufactured by Qiagen
The SiBRCA2 is a laboratory equipment product designed for DNA analysis. It is used to detect and quantify the expression of the BRCA2 gene, which is associated with hereditary breast and ovarian cancer. The SiBRCA2 provides a reliable and efficient method for researchers to study the BRCA2 gene in their scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Sibrca1, by Horizon Discovery (1 mentions)
Dulbecco s modified eagle medium, by Lonza (1 mentions)
Sigenome brca2 smartpool, by Horizon Discovery (1 mentions)
Sitp53, by Horizon Discovery (1 mentions)
On targetplus non targeting pool sirna, by Horizon Discovery (1 mentions)
Dharmafect 1, by Horizon Discovery (1 mentions)
Interferin, by Polyplus Transfection (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Flp in 293, by Thermo Fisher Scientific (1 mentions)
Sirna id s4219, by Thermo Fisher Scientific (1 mentions)
3 protocols using sibrca2
1
Cell Culture and Transfection Protocol
2
siRNA-mediated knockdown of DNA repair genes
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Cells were transfected with siRNAs using Dharmafect1 (Horizon Discovery) or INTERFERin (Polyplus) according to manufacturer’s instructions. Briefly, cells were transfected with 40 nM siRNAs by reverse transfection. The next day, the medium was replaced by fresh medium. The following siRNAs were used: siBRCA2 (Qiagen, SI02653434), siGENOME BRCA2 SMARTpool (Dharmacon, M-003462-01), siTP53 (Dharmacon, GCA UCU UAU CCG AGU GGA AUU UU), siRAD52 SMARTpool (Dharmacon, L-011760-00-0005). AllStars Negative Control siRNA (Qiagen, 1027281) or ON-TARGETplus non-targeting pool siRNA (Dharmacon, D-001810-10-05) were used as control.
3
DART System with TRDMT1 Knockout U2OS Cells
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U2OS TRE40 (link), TRDMT1 KO U2OS TRE22 (link), Flp-in 293 (Thermo, Cat#R75007), HS578T and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Cat#12–604 F, Lonza; Basel, Switzerland) with 10% (vol/vol) FBS at 37 °C with 5% CO2. The U2OS-TRE cells used for the DART system have been described in previous articles40 (link). The HS578T and MDA-MB-231 cells are gifts from Dr. Leif W. Ellisen. For plasmid and siRNA transfection, Lipofectamine 2000 and Lipofectamine RNAiMax (Invitrogen; Carlsbad, CA, USA) were used following the manufacturer’s standard protocol, respectively. The siRNA for TRDMT1 was purchased from Invitrogen (siRNA ID: s4219, Cat#: 4392420). Other siRNAs include siBRCA1 (L-003461-00, Dharmacon), and siBRCA2 (GS675, Qiagen).
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