shRNA clones targeting each of Fbxl19 and Rnf20 were purchased from Sigma-Aldrich (Supplemental Table S1 ). Lentiviruses expressing a specific shRNA were generated by transfecting 6 μg of pLKO vector containing a specific shRNA, 4 μg of Δ8.9, and 2 μg of VSVG onto 9 × 106 293T cells using the Fugene transfection reagent (Promega). The following day, the medium was replaced with ES cell culture medium (or LIF-medium for differentiation study), and then incubated at 37°C for 24 h. Viral supernatant was collected, filtered with a syringe filter (0.45 μm), and used to infect ES cells. To knockdown Fbxl19 or Rnf20, 2.5 × 105 J1 ES cells in each well of a 24-well plate were infected with viral particles. Infected cells were cultured overnight, and the medium was replaced by fresh culture medium supplemented with puromycin (1 μg/ml) to select for infected cells. To investigate acute expression changes upon KD, cells were harvested within 48 h after infection. For gene expression analysis, cells were harvested after 4 days of infection.
Shrna clones
Manufactured by Merck Group
Sourced in United States
ShRNA clones are a type of laboratory equipment used for gene silencing experiments. They contain short hairpin RNA (shRNA) sequences that can target and suppress the expression of specific genes in cell lines and model organisms. ShRNA clones provide a tool for researchers to investigate the function of genes and their role in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Fugene transfection reagent, by Promega (1 mentions)
Jetprime transfection reagent, by Polyplus Transfection (1 mentions)
Sirnas, by Bioneer (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Roche (1 mentions)
Mission lentiviral packaging mix, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Plenti giii cmv gfp 2a puro backbone, by Applied Biological Materials (1 mentions)
Puromycin, by AG Scientific (1 mentions)
8 protocols using shrna clones
1
Lentiviral shRNA Knockdown in Embryonic Stem Cells
2
TRIM65 Gene Silencing Methodology
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shRNA clones targeting mRNA of TRIM65 were from Sigma-Aldrich. Sequences of shRNA are as follows: shTRIM65 (1), 5′-GAATTATCGCAATCTGACCTT-3′; and shTRIM65 (2), 5′-CCGTCCTGTCTTGTAGTCTTT-3′. shTRIM65 (1) targets the coding region of TRIM65 gene, and shTRIM65 (2) targets the 3′ UTR region of TRIM65 gene. The protocol for generating HeLa cells stably expressing shRNA has been published (Yan et al., 2013 (link)).
3
Transient and Stable Knockdown of Genes
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For the transient knockdown of α7nAChR, SRC, AGT, and STAT3, the cells were transfected with scrambled or gene-specific small interfering RNAs (siRNAs, purchased from Bioneer (Daejeon, Republic of Korea)) using the JetPRIME transfection reagent (Polyplus-transfection SA, Illkirch, France) according to the manufacturer’s instructions. Gene knockdown was confirmed with Western blotting or real-time polymerase chain reaction (PCR). For generation of stable knockdown cell lines with reduced AGTR1 or IGF2R expression, 1170-I, 1799, or BEAS-2B cells were transduced with lentiviral particles with shRNA clones against AGTR1 or IGF2R (Sigma‒Aldrich), followed by selection with puromycin.
4
Lentiviral Knockdown of Human UGT1
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Four shRNA clones targeting human UGT1 and the pLKO.1 control plasmid were purchased through TRC consortium from Sigma. To generate lentivirus, 3 μg of shRNA clone, 3 μg of pCMV8.2ΔR, and 1.5 μg VSV-G expression plasmid were transfected into 293T cells in 6-cm plates by lipofectamine 2000 (Invitrogen). Viruses were collected at 48 hours post-transfection and cleared through filtration. 500 μl viruses were added to Huh7.5.1 cells in a 12-well plate and selected by puromycin (0.6 μg/ml). To verify the knockdown efficiency, cell lysates were prepared and analyzed by Western blotting.
5
TWIST1 Expression Modulation in Cells
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For TWIST1 overexpression, lentiviral plasmids encoding full‐length wide‐type TWIST1 with a pLenti‐GIII‐CMV‐GFP‐2A‐Puro backbone (Applied Biological Materials Inc., Vancouver, BC, Canada) were used. For TWIST1 knockdown, two shRNA clones (#TRCN0000020541 and #TRCN0000020542; Sigma‐Aldrich; subsidiary of Merck KGaA: St. Louis, MO, USA) were selected with pLKO.1‐puro Luciferase shRNA plasmid (#SHC007; Sigma‐Aldrich) as a control. Plasmids were mixed with MISSION® Lentiviral Packaging Mix (#SHP001; Sigma‐Aldrich) before added to a mixture of transfection reagent Fugene 6 (#11814443001; Roche, Basel, Switzerland) and OptiMEM. After 10–15 min’ incubation at room temperature, they were added to 293T cells seeded in the 6‐cm dishes. For infection, virus‐containing supernatants were harvested 48 and 72 h after transfection, filtered, and added to selected cells, together with polybrene (Sigma‐Aldrich). Twenty‐four hours after infection, cells were treated with puromycin at a proper concentration decided by their respective puromycin kill curve.
6
HeLa Cell Transfection and Clonal Selection
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HeLa cells were seeded at 50–70% confluency and transfected with ShRNA clones obtained from Sigma Aldrich (USA). Stably transfected cells were selected using DMEM supplemented with 2 μg/ml puromycin. These cells, termed as Sh, were further subjected to clonal selection and three clones-Sh1, Sh2, and Sh3 were obtained.
7
Lentiviral shRNA-Mediated Transcription Factor Knockdown
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shRNA clones targeting each of TFs were purchased from Sigma-Aldrich (Supplementary Data 11 ). Lentiviruses expressing a specific shRNA were generated by transfecting 6 µg of pLKO vector containing a specific shRNA against a gene of interest, 4 µg of Δ8.9, and 2 µg of VSVG into 9 × 106 293T cells. After 12 h, the medium was replaced with the TSC culture medium and then incubated at 37 °C for 36 h. Viral supernatant was collected, filtered with a syringe filter (0.45 µm), and used to infect TSCs. To knock down each factor, 1 × 105 TSCs in each well of a 12-well plate were infected with viral particles. Infected cells were cultured for 24 h, and the medium was replaced by fresh TSC culture medium with puromycin (1 μg/ml) for the selection of infected cells. For gene expression analysis, cells were harvested after 3 days of infection. For differentiation of TSCs upon KD of each factor, the culture medium was replaced with TS basal medium without Fgf4 and heparin after 3 days of infection.
8
Downregulation of HS and CS Synthesis
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Heparan sulfate (HS) and chondroitin sulfate (CS) are saccharides; thus, they could not be downregulated directly by short hairpin RNAs (shRNAs) but rather by targeting key enzymes involved in their synthesis, namely, exostosin glycosyltransferase (EXT) and chondroitin sulfate synthase (CHSY), respectively. Down-regulation of EXT or CHSY expression was performed by lentiviral infection with shRNA. Several different shRNAs were assayed and the most effective ones were chosen for use throughout the study. In the case of EXT, down-regulation of EXT2 was performed by a combination of two different shRNA clones (both from Sigma-Aldrich, Rehovot, Israel): 1) Cat. No. SHCLNG-NM_000401.1-2839s1c1 and 2) Cat. No. SHCLNG-NM_000401.x-2366s1c1. In the case of CHSY, down-regulation of CHSY1 was assumed by shRNA Cat. No. SHCLNG-NM_014918.3-1964s1c1 (Sigma-Aldrich). Following infection, selection was performed with 4 μg/ml puromycin (A.G. Scientific, San Diego, CA). Then, the cell population was used as a whole to prevent bias toward specific cell clones. Control cells were infected with control shRNA vectors (Sigma-Aldrich) carrying similar antibiotic resistance. Down-regulation of EXT2 or CHSY1 was verified by reduced expression of HS or CS in the cells, using confocal analyses (please see below).
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