Sigenome smartpool

Manufactured by Horizon Discovery

Sourced in United States, United Kingdom

The SiGENOME SMARTpool is a pre-designed and optimized pool of short interfering RNA (siRNA) sequences targeting a specific gene. It is designed to provide effective gene silencing with reduced off-target effects.

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SMARTpool (292 citations) SiGENOME SMARTpool siRNA (134 citations) SiGENOME Human SMARTpool (8 citations) SiGENOME SMARTpool siRNA reagents (6 citations) SiGENOME siRNA SMARTpool® reagents (4 citations) SiGENOME SMARTpool XBP1 (4 citations) SiGENOME SMARTpool siRNA library (4 citations) SiGENOME SMARTpools library (2 citations) SiGENOME SMARTpool RNF31 (2 citations) SiGENOME BRCA2 SMARTpool (2 citations)

203 protocols using sigenome smartpool

siRNA Transfection Protocol for Functional Genomics

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siRNAs were incubated with Lipofectamine 2000 and serum-free Optimem for 15 minutes at room temperature. Cells were then trypsinized and resuspended in 1 ml of medium and plated directly into the siRNA/Optimem/Lipofectamine solution at 50% confluence and incubated for 48 hours. Sequences of siRNA oligonucleotides used here are as follows: Control non-targeting siRNA, 5′-UAGCGACUAAACACAUCAA-3′ (Dharmacon); HORMAD1 siRNA #1, 5′-GGA CAA AGA UGU AGA AGA U-3′, HORMAD1 siRNA #2, Dharmacon siGenome smart pool cat# M-018596-02; BRCA1, Dharmacon siGenome smart pool cat# M-003461-02; BRCH1, Dharmacon siGenome smart pool cat# M-010587-00; Lig4, Dharmacon siGenome smart pool cat# M-004254-00. For HORMAD1 depletions siRNA#1 was used unless otherwise indicated.

Gao Y., Kardos J., Yang Y., Tamir T.Y., Mutter-Rottmayer E., Weissman B., Major M.B., Kim W.Y, & Vaziri C. (2018). The Cancer/Testes (CT) Antigen HORMAD1 promotes Homologous Recombinational DNA Repair and Radioresistance in Lung adenocarcinoma cells. Scientific Reports, 8, 15304.

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Silencing Transcription Factors in BMDMs

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Egr2 interfering RNAs (siRNAs) (siGenome smart pool, cat# M-040303-01), and CEBPβ siRNA (siGenome smart pool, Cat # M-043110-02), and CEBPα siRNA [(siGenome smart pool, Cat # M-040561-01) and control siRNA (siGenome non-targeting control pool), cat# D-0011206-13-05] were purchased from Dharmacon (Lafayette, CO, USA) and used similarly as described earlier in our studies (36 (link)). BMDMs were transfected with siRNAs using TransIT-X2 System as a transfecting agent (Madison, WI, USA) according to the manufacturer's instructions. The efficiency of transfection was 80–90% as determined by the expression of a fluorescent reporter (Cy3-labeled RNA) in F4/80-positive cells as determined by flow cytometry.

Veremeyko T., Yung A.W., Anthony D.C., Strekalova T, & Ponomarev E.D. (2018). Early Growth Response Gene-2 Is Essential for M1 and M2 Macrophage Activation and Plasticity by Modulation of the Transcription Factor CEBPβ. Frontiers in Immunology, 9, 2515.

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siRNA Knockdown of p63, ACC1, and HER2

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LX-2 cells were transfected with specific small-interference RNA (siRNA) to knockdown the expression of total p63 (siGENOME SMARTPool, L-003330-00-0005, Dharmacon), ACC1 (siGENOME SMARTPool, L-004551-00-0005, Dharmacon) or HER2 (siGENOME SMARTPool, L-003126-00-0005, Dharmacon). Control group was administered with a non-targeting siRNA (siGENOME Non-Targeting siRNA Pool, D-001206-13-05, Dhamacon). The transfection was performed using Lipofectamine 2000 (11668-019, Invitrogen). Briefly, cells were seeded in 6-wells plates. Then, 0.05 nmol of each siRNA diluted in 300μL of optiMEM (31985070, Life Technologies) was mixed with 6 μL of Lipofectamine 2000 diluted in 294 μL of optiMEM; the mixture was added into each well, resulting in a final volume of 600 μL. The medium was replaced with fresh medium after 6 h, and cells were collected after a total of 48 h to check the efficiency of silencing by Real Time PCR.

Fondevila M.F., Novoa E., Gonzalez-Rellan M.J., Fernandez U., Heras V., Porteiro B., Parracho T., Dorta V., Riobello C., da Silva Lima N., Seoane S., Garcia-Vence M., Chantada-Vazquez M.P., Bravo S.B., Senra A., Leiva M., Marcos M., Sabio G., Perez-Fernandez R., Dieguez C., Prevot V., Schwaninger M., Woodhoo A., Martinez-Chantar M.L., Schwabe R., Cubero F.J., Varela-Rey M., Crespo J., Iruzubieta P, & Nogueiras R. (2024). p63 controls metabolic activation of hepatic stellate cells and fibrosis via an HER2-ACC1 pathway. Cell Reports Medicine, 5(2), 101401.

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High-throughput siRNA Screening in CDK12 Mutant Cells

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CDK12^as cells (1000 cells/well) were reverse transfected in a 96 well plate format with a custom siGENOME SMARTPool (Dharmacon) siRNA library as described previously^{72 (link)} using Lipofectamine RNAimax Transfection Reagent (Promega). Positive (siPLK1) and negative controls (siCON1, Dharmacon) were also included in each plate. After 24 hours, media was replaced with new media drug containing 1NM (0.3 μM) or the drug vehicle (DMSO), and cells were continuously cultured for six days further, at which point cell viability was estimated by the addition of CellTiter-glo reagent to the media for 10 minutes. Drug Effect Z scores were calculated from the resultant luminescence data as described previously^{73 (link)}. Each screen was carried out in triplicate, with the data being combined in the final analysis. For single gene siRNA experiments, C4-2B cells were plated in 96-well plates and allowed to adhere overnight. The next day, cells were transfected using siGENOME SMARTPool (Dharmacon) against the indicated genes (CCNK, CDK13) or nontargeting control (NTC) as above. The plate was then placed in an IncuCyte S3 (Sartorius) and cell growth monitored over the indicated time frame.

Tien J.C., Chang Y., Zhang Y., Chou J., Cheng Y., Wang X., Yang J., Mannan R., Shah P., Wang X.M., Todd A.J., Eyunni S., Cheng C., Rebernick R.J., Xiao L., Bao Y., Neiswender J., Brough R., Pettitt S.J., Cao X., Miner S.J., Zhou L., Wu Y.M., Labanca E., Wang Y., Parolia A., Cieslik M., Robinson D.R., Wang Z., Feng F.Y., Lord C.J., Ding K, & Chinnaiyan A.M. (2024). CDK12 Loss Promotes Prostate Cancer Development While Exposing Vulnerabilities to Paralog-Based Synthetic Lethality. bioRxiv.

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Transfection of miRNA and siRNA

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The miRVana miR-634 mimic (4464066) and negative control 1 (miR-NC; 4464058), anti-miR-634 (AM17000), and anti-miR-NC (AM17010) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The siRNA targeting ASCT2 (pool of four sequences; siGENOME SMARTpool; M-007429-01) and negative control (siNC; siGENOME SMARTpool; D-001206-14) were obtained from Dharmacon (Lafayette, CO, USA). Cells were transfected with miRNA or siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Inoue J., Fujiwara K., Hamamoto H., Kobayashi K, & Inazawa J. (2020). Improving the Efficacy of EGFR Inhibitors by Topical Treatment of Cutaneous Squamous Cell Carcinoma with miR-634 Ointment. Molecular Therapy Oncolytics, 19, 294-307.

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Antibody Validation and siRNA Knockdown for DNA Repair Proteins

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The following antibodies were used in this study at the indicated dilutions: anti-CIP2A (CST #14805; 1 : 1000), anti-GAPDH (Sigma-Aldrich G9545; 1 : 20 000), anti-GFP (gift from Laurence Pelletier, 1 : 10 000), anti-KAP1 (Bethyl A300-274A, 1 : 5000), anti-POLE3 (Bethyl A301-245A-1; 1 : 2000), anti-POLE4 (Abcam ab220695; 1 : 200), anti-Tubulin (Millipore CP06, 1 : 2000), anti-pCHK1 (S345) (Cell Signaling #2348, 1 : 1000), anti-CHK1 (Santa Cruz sc8408, 1 : 500), anti-pRPA32 (S33) (Bethyl A300-246A-3, 1 : 20 000), anti-RPA32 (Abcam ab2175, 1 : 500). The following secondary antibodies for immunoblotting were used in this study: peroxidase-conjugated AffiniPure Bovine Anti-Goat IgG (Jackson Immuno Research 805-035-180) and peroxidase-conjugated Sheep Anti Mouse IgG (GE Healthcare NA931 V). All peroxidase-conjugated secondary antibodies were used at a dilution of 1 : 5000. Protein bands were detected using the SuperSignal West Pico enhanced chemiluminescence reagent (Thermo Fisher Scientific). The following siRNAs from Dharmacon were used in this study: control, siGENOME Non-targeting Pool #2 (D-001206-14-05); POLE3, siGENOME SMARTpool (M-008460-01-0005); POLE4, siGENOME SMARTpool (M-009850-01-0005); APEX2, siGENOME SMARTpool (M-013730-00-0005). ATR inhibitors VE-821 and AZD6738 were purchased from SelleckChem.

Hustedt N., Álvarez-Quilón A., McEwan A., Yuan J.Y., Cho T., Koob L., Hart T, & Durocher D. (2019). A consensus set of genetic vulnerabilities to ATR inhibition. Open Biology, 9(9), 190156.

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RNAi-mediated Knockdown of Nuclear Proteins

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siRNAs targeting SFPQ (siGENOME SMARTpool, [M-006455-02]) and PSPC1 (siGENOME SMARTpool, [M-020596-01]) were purchased from GE Healthcare Dharmacon Inc. siRNAs targeting NEAT1 (Silencer Select Human NEAT1 siRNA, [4390771]; Ambion) and negative control (Silencer Select Negative Control No. 1 siRNA, [4390844]; Ambion) were also used. siRNA transfections of HeLa^GFP-NONO in the RNAi treatments were performed with Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. Briefly, for a 12-well format (100,000 cells were seeded per well), 2 μl of Lipofectamine 2000 was diluted in 100 μl of optiMEM medium (Gibco). About 40 pmol of each siRNA was then diluted in 100 μl of optiMEM medium and incubated at RT for 5 min. The diluted Lipofectamine 2000 and diluted siRNA were then mixed and incubated at RT for 30 min. siRNA-Lipofectamine complexes were then directly added to seeded cells, mixed gently, and incubated at 37 °C for approximately 4 h; siRNA-Lipofectamine complexes were then removed and replaced with fresh HeLa media.

Lee P.W., Marshall A.C., Knott G.J., Kobelke S., Martelotto L., Cho E., McMillan P.J., Lee M., Bond C.S, & Fox A.H. (2022). Paraspeckle subnuclear bodies depend on dynamic heterodimerisation of DBHS RNA-binding proteins via their structured domains. The Journal of Biological Chemistry, 298(11), 102563.

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siRNA Screening Workflow in HeLa Cells

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For siRNA screening, HeLa cells cultured in 96-well glass-bottom plates (IWAKI) were treated with siGENOME SMARTpool, which included four unique siRNAs designed for one target gene (Dharmacon). Cells were incubated with siRNA using RNAiMAX (Invitrogen) for 48 h. To evaluate the RNAi knockdown efficiency, each 96-well plate contained a well for a non-targeting siRNA with the sequence CUUACGCUGAGUACUUCGA (GL3, Nippon EGT) as the negative control. The plate also contained a well for the positive control, siLMNA (Dharmacon siGENOME SMARTpool: M-004978-01-0005), which was immunostained with anti-LaminA/C under the same conditions used for the other siRNAs. The other SMARTpool siRNAs (Dharmacon) used in this study are as follows:
RPL5: M-013611-01-0005 and L-013611-00-0005, TYMP: M-009281-01-0005, POP4: M-020046-00-0005, RPL9: M-011139-01-0005, RPL13: M-013714-00-0005, RPS9: M-011131-01-0005, and TIF-IA(RRN3): L-016947-00-0005. The individual siRNAs (Dharmacon) used in this study are as follows:

RPL5 #1: GACAAACAGAGAUAUCAUU, RPL5 #2: GAUGAUAGUUCGUGUGACA

RPL21 #1: J-012910-07-0002, and RPL21 #2: J-012910-05-0002

Matsumori H., Watanabe K., Tachiwana H., Fujita T., Ito Y., Tokunaga M., Sakata-Sogawa K., Osakada H., Haraguchi T., Awazu A., Ochiai H., Sakata Y., Ochiai K., Toki T., Ito E., Goldberg I.G., Tokunaga K., Nakao M, & Saitoh N. (2022). Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly. Life Science Alliance, 5(7), e202101045.

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siRNA Knockdown of p62/SQSTM1

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The siRNAs for p62/SQSTM1 (siGENOME SMARTpool; M-010230-00-0005) and nontargeting negative control (siGENOME SMARTpool; D-001206-14-05) were obtained from GE Dharmacon (Pittsburgh, PA). The cells were transfected with 10 nmol/L of each siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions.

Iwadate R., Inoue J., Tsuda H., Takano M., Furuya K., Hirasawa A., Aoki D, & Inazawa J. (2015). High Expression of p62 Protein Is Associated with Poor Prognosis and Aggressive Phenotypes in Endometrial Cancer. The American journal of pathology, 185(9).

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RNA Interference of Endocytic Proteins

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5×10⁵ RAW 264.7 cells were cultured overnight in 6-well plates in DMEM supplemented with 10% FCS. Prior to transfection, cells were washed three times with 1X PBS and replenished with DMEM containing exosome-depleted FCS, produced by spinning the FCS at 100,000×g for 1 hour to remove exosomes. The transfection mix consisted of 75nM of target siRNA in 100uL of DMEM containing 10% HiPerfect (Qiagen). siRNA for Tsg101, Hrs, Park2 and control used were siGENOME SMARTpools (Dharmacon). The oligonucleotides used for the knockdowns are as follows. Tsg101 primers: 5′-CCGCUUAGAUCAAGAAGUAUU-3′, 5′-CGUAAACAGUUCCAGCUAAUU-3′, 5′-UACAAUCCCAGUGCGUUAUU-3′, 5′-UGUCAUCGCUAUGUACAAAUU-3′. Hrs primers: 5′-AGAGACAAGUGGAGGUAA-3′, 5′-UUUACCUCCACUUGUCUC-3′, 5′-GCACGUCUUUCCAGAAUUCAA-3′, 5′-ACAAGAACCCACACGUC-3′. Parkin primers: 5′-GGACUACAUGAUUCGACGUCAACUG-3′, 5′-GGAAACAUCAGUAGCUUUGCACCUG-3′, 5′-UUGCUUAGACUGUUUCCACUUAUAC-3′, 5′-GAUGACUAAACCUGACAGAA-3′. Knockdown was confirmed by western blot using 10ug of whole cell lysate.

Smith V.L., Jackson L, & Schorey J.S. (2015). Ubiquitination as a mechanism to transport soluble mycobacterial and eukaryotic proteins to exosomes. Journal of immunology (Baltimore, Md. : 1950), 195(6), 2722-2730.

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