pEGFP-neprilysin-Neo/kanamycin-resistant gene cDNA clone plasmid was purchased from Cosmogenetech (Seoul, Korea). The plasmid DNA containing the kanamycin resistance gene, NEP, and green fluorescent protein (GFP) gene was transformed into HIT competent cell T-DH5a (RBC Bioscience, New Taipei, Taiwan). Competent cells were proliferated in Bacto agar medium (BD Difco™, Bergen County, NJ, USA) and LB broth (BD Difco™, Bergen County, NJ, USA) containing kanamycin. The transformation plasmid DNA was extracted using the NucleoBond Xtra Midi Plus Kit (Macherey-Nagel, Bethlehem Township, PA, USA). The extracted plasmid DNA was reacted with Lipofectamine Stem Reagent (STEM00015, Invitrogen, USA) and Opti-MEM™ I Reduced Serum Medium (Gibco, Gaithersburg, MD, USA) and then added to the culture medium in which hUC-MSCs grew. To confirm the transfection of cells after 2 days, the level of GFP was observed using a Carl Zeiss Axiovert 200M microscope (Carl Zeiss, Baden-Württemberg, Germany).
Lipofectamine stem reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine Stem Reagent is a transfection reagent designed for efficient delivery of nucleic acids into stem cells and other difficult-to-transfect cell types. It facilitates the uptake of DNA, RNA, and other macromolecules into the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions)
Dual luciferase reporter assay system, by Promega (4 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Pgl3 vector, by Promega (2 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
T4 ligation, by New England Biolabs (2 mentions)
Quick ligation, by New England Biolabs (2 mentions)
Pspcas9 bb 2a puro px459 v2, by Addgene (2 mentions)
Anti caspase 8 primary antibody, by Cell Signaling Technology (2 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (2 mentions)
Lenticrispr v2, by Addgene (2 mentions)
Murine ifn γ, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Cold fusion cloning kit, by System Biosciences (2 mentions)
Nucleobond xtra midi plus kit, by Macherey-Nagel (1 mentions)
32 protocols using lipofectamine stem reagent
1
Transfection of hUC-MSCs with pEGFP-neprilysin-Neo
2
Regulatory Region R5 Luciferase Assay
3
Generation of Inducible Overexpression Cell Lines
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SRSF2, SF3A3, SNRPD1, SNRPE, NUMB‐S, and HES1 cDNAs were amplified from H1 cDNA or purchased from company (BGI, China). They were cloned into the tet‐on inducible piggybac transposon expression vector (Yusa et al,2011 ), co‐expressing the GFP (P2A GFP) and possessing puromycin resistance. The plasmids were co‐transfected with transposase to H1 with the Lipofectamine Stem reagent (cat# L3000015, Invitrogen) following the manufacturer’s instructions. Twenty‐four hours after transfection, 1 μg/ml puromycin was added to the culture media for selection for approximately 10 days until nearly no dead cells observed. The cells were induced by 1 μg/ml DOX for 2.5 days to assess GFP expression. The GFP+ cells reaching ~ 90% were referred as stable cell lines, and the expression of target genes was measured by Western blotting. The primers used are listed in Table EV1 .
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SRSF2, SF3A3, SNRPD1, SNRPE, NUMB‐S, and HES1 cDNAs were amplified from H1 cDNA or purchased from company (BGI, China). They were cloned into the tet‐on inducible piggybac transposon expression vector (Yusa et al,
4
High-Res Microscopy of Organelle Dynamics
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The cells were split as colonies using ReLeSR and seeded in chambered coverglass for high-resolution microscopy (Thermo Scientific, Nunc™ Lab-Tek™ II Chambered Coverglass #1.5, 155379). Transfection was done one-day post-splitting. Approximately 20 min before transfection, media was replaced with Essential 8 (Gibco, A1517001). The transfection mix was prepared in Opti-MEM 1X (Gibco 31985070) with 0.5 μl of Lipofectamine Stem Reagent (Invitrogen, STEM00001), and 500 ng of total DNA divided as follows: 167 ng of pEIF1a::Transposase (gifted by Dr. Michael Ward), and 333 ng of organelle markers: lysosomes [pEIF1a::LAMP1::mTurquoise], mitochondria [pEIF1a::Cox8(1-26)::eGFP], Golgi [pEIF1a::Sit::OxVenus], peroxisomes [pEIF1a::mOrange2::SKL], endoplasmic reticulum [pEIF1a::Sec61β::mApple] (Twist Technologies). The mix was applied dropwise and cells were incubated for four hours before the media was changed back to StemFlex and supplemented with BODIPY™ 665/676 (80 ng) for the staining of lipids (Invitrogen, B3932). Prior to imaging, a media change was performed.
5
Knockdown of IGF2 in Cell Cultures
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Lipofectamine Stem Reagent (Invitrogen) and Opti-MEM I reduced serum medium (Invitrogen) were used for the transfection experiments according to the manufacturer’s specifications. 1 × 106 cells were transfected with 2 µM IGF2 (h) siRNA (Santa Cruz Biotechnology) or Silencer® Select Negative Control No. 2 siRNA (Invitrogen). After 24 h, the medium was replaced with fresh medium and incubated for another 24 h, before cells were harvested and expression upon knockdown of interest was analyzed using quantitative real-time PCR.
6
Modulating circZNF91 in Leukemia Cells
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MEC-1 cells were cultured with RPMI-1640 medium, and the HG-3 cells were cultured in DMEM medium, supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). To investigate the role of circZNF91, MEC-1 cells and HG-3 cells were transfected with 100 nM siRNA targeting circZNF91. For miRNA, cells were transfected with 50 nM of mimics and controls or inhibitors. Transfection was performed with Lipofectamine™ Stem Reagent (STEM00001, Invitrogen). In a 6-well plate, 60% confluent cells were transfected with 50 nM of a microRNA mimic/inhibitor, 2 μg of plasmid, or 100 nM siRNA, according to the manufacturer's instructions. Transfected cells were subjected to subsequent analysis 48 h posttransfection.
7
LNA-mediated Knockdown of HERVH
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The design of LNA oligonucleotides was based on the study by Wang et al (2014a) (link) in which three sequences of shRNAs (#3, #4 and #12) targeting HERVH were shown to have effective knockdown efficiency (Wang et al, 2014a (link)). HERVH LNA oligonucleotides were ordered from Exiqon (QIAGEN). For each well of a 24-well plate, 1 × 105 cells were seeded on Geltrex coating with 1 ml mTesR1 medium containing 10 μM RCOK inhibitor. Cells were placed in an incubator with 5% CO2 at 37°C for 2–4 h to allow them to re-attach to the plate before transfection. LNA-GapmeRs were prepared with nuclease-free water at 50 μM stock concentration. Lipofectamine Stem Reagent (Invitrogen) was warmed to reach room temperature before use. Per reaction, 100 pmol in total of LNA oligonucleotide(s) and 3.5 μl of Lipofectamine Stem Reagent were added to 100 μl Opti-MEM. The LNA and Lipofectamine Stem Reagent containing tubes were mixed and incubated at RT for 10 min. LNA-Lipofectamine Stem Reagent mixture was added to seeded cells with fresh mTesR1 medium. The medium was gently mixed and cells were returned to the incubator. Cells were harvested after 24-h treatment either for RNA extraction or for CUT&RUN protocols.
8
Sp1 Overexpression in HC11 Cells
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HC11 cells were grown to 70% confluence and washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco), then changed into Opti-MEM (Gibco) medium for 2 h. siRNA or plasmids were transfected using Lipofectamine stem reagent (Invitrogen, CA, United States) according to the manufacturer’s instructions. After 24–48 h of transfection, the cells were collected for further experiments. The pcDNA 3.1 empty vector and pcDNA 3.1-Sp1 overexpression vector were purchased from GenePharma company (Suzhou, China). The siRNA sequences and shBmpr1a target sequences are listed in Supplementary Table 2 .
9
Generating USP3 Knockout Cell Lines
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To generate USP3 knockout single-cell-derived clones, we co-transfected NCCIT or hESCs with the plasmids encoding Cas9 and sgRNA targeting USP3 or non-targeted sgRNAs (scrambled sgRNAs) at a 1:2 ratio using polyethyleneimine in NCCIT (PEI; Polysciences, Warrington, PA, USA) or Lipofectamine STEM reagent in hESCs (STEM00003, Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Transfected cells were sorted by FACS and reseeded into 24-well plates for recovery. Cells were dissociated and seeded again into 96-well plates to establish single-cell-derived colonies. After 15 days, single-round colonies were marked and expanded for screening by the T7E1 assay. USP3 KO-positive clones were confirmed by T7E1 and Sanger sequencing. Scrambled sgRNA-targeted cells that are T7E1-negative and showing no gene disruption of USP3 gene were used as controls for all the experiments.
T7E1-positive USP3 KO hESC clones #2, #5, and #9 generated by single-cell dilution were used for further characterization experiments described inFigure 5 D–I.
T7E1-positive USP3 KO hESC clones #2, #5, and #9 generated by single-cell dilution were used for further characterization experiments described in
10
Luciferase Reporter Assay for Transcriptional Regulation
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For reporter assay, the promoter regions of candidates were cloned into pGL3 vector (Promega). The primers for generating these reporters are listed in Table S3 . The RHOXF1- and RHOXF2-shRNA plasmids were generated by inserting ologonucleotides corresponding to siRNA sequences (Table S3 ) into the pLLU2G backbone. HEK293T cells were transfected with the Lipofectamine 2000 reagent (Invitrogen). GC1 and TCam-2 cells were transfected with the Lipofectamine 3000 reagent (Invitrogen). GS cells were transfected with the Lipofectamine stem reagent (Invitrogen). For transfection experiments, most cells were trypsinized and seeded in 24-well plates at a density of ~70,000 cells per well. TCam-2 cells were seeded at 20,000 cells/well. Transfection was performed following the manufacturer’s instructions. For lentiviral transduction, 1 infectious unit (IU) of lentivirus per cell was added with 6 μg/ml polybrene. Cells were harvested 1 day post-transfection for luciferase activity analysis. Luminescence was measured using the Dual-Luciferase Reporter assay system (Promega) following the manufacturer’s instruction. The pRL-cmv (Renilla) vector was co-transfected in these experiments as an internal control for normalization. Statistical significance was determined using the paired Student’s t-test.
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