Linear circ-HuR and hsa_circ_23897 were synthesized by TSINGKE (Wuhan, China) and inserted into pLCDH-ciR (Geenseed Biotech Co., Guangzhou, China). Mutation of back-splicing elements of circ-HuR or hsa_circ_23897 vector was prepared with GeneTailor™ Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA) and primers (Additional file 1 : Table S3). Human CNBP cDNA (540 bp) was subcloned into CV186 (Genechem Co., Ltd., Shanghai, China), while its truncations were obtained by PCR amplification (Additional file 1 : Table S3) and inserted into pCMV-3Tag-1A or pGEX-6P-1 (Addgene, Cambridge, MA, USA), respectively. Two independent single guide RNAs (sgRNAs) targeting downstream region of CNBP transcription start site (Additional file 1 : Table S4) were inserted into dCas9-BFP-KRAB (Addgene). Oligonucleotides specific for short hairpin RNAs (shRNAs) against circ-HuR (Additional file 1 : Table S4) were inserted into GV298 (Genechem Co., Ltd). Lentiviral vectors were co-transfected with packaging plasmids psPAX2 and pMD2G into HEK293T cells. Infectious lentiviruses were harvested at 36 and 60 h after transfection, followed with concentration by ultracentrifugation (2 h at 120,000 g). Stable cell lines were obtained by selection with puromycin.
Gv298
Manufactured by Genechem
Sourced in China
The GV298 is a laboratory centrifuge designed for general-purpose applications. It features a compact and durable construction, allowing for efficient separation of various samples. The centrifuge can accommodate a range of rotor configurations and has adjustable speed and time settings to cater to different experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Dcas9 bfp krab, by Addgene (5 mentions)
Puromycin, by Thermo Fisher Scientific (5 mentions)
Neomycin, by Thermo Fisher Scientific (5 mentions)
Pcmv 3tag 1a, by Addgene (4 mentions)
Genetailor site directed mutagenesis system, by Thermo Fisher Scientific (4 mentions)
Pgex 6p 1, by Addgene (4 mentions)
Cv186, by Genechem (3 mentions)
Pcmv n myc, by Addgene (3 mentions)
Pcdna3, by Thermo Fisher Scientific (3 mentions)
Pcmv 3tag 1c, by Addgene (3 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Gv141, by Genechem (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dcas9 vpr, by Addgene (1 mentions)
Pegfp n1, by Addgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Plcdh cir, by Genechem (1 mentions)
Cv186 lentivirus vector, by Genechem (1 mentions)
Pcmv ha, by Addgene (1 mentions)
Pet28a, by Addgene (1 mentions)
13 protocols using gv298
1
Circular RNA Manipulation and Expression
2
Establishing GLUT4-Overexpressing Cell Lines
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The Flag‐tagged GLUT4 in eukaryotic expression vector GV141 was purchased from Genechem (Shanghai, China). BGC823 cells were transfected with GLUT4 plasmids or control plasmids followed by 1 μg/mL purine (Gibco) screening for 2 weeks to obtain stable GLUT4‐overexpressing or control cells. Lentiviral constructs pLenti6 were purchased from Invitrogen (Carlsbad, CA, USA). shRNA constructs were constructed by cloning shRNA fragments into pLenti‐U6 (Invitrogen) and GV298 (Genechem). The sequences of shRNAs for ISL1 and GLUT4 knockdown are listed in Supplementary Table S1 .
Stable cell lines were established with a lentiviral vector using previously described protocols [32 ]. Briefly, lentiviruses were used to infect the cells for 2 days. Stable clones were selected by treating the cells with 3 μg/mL blasticidin (Gibco) for 2 weeks.
Stable cell lines were established with a lentiviral vector using previously described protocols [
3
Generating circNR3C1 Plasmid and Ectopic Vectors
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The human circNR3C1 plasmid was constructed and used as our previous study described.25 (link) The C-myc and EZH2 promoter luciferase reporter vectors, ectopic vectors targeting BRD4, C-myc, and EZH2 were synthesized in our previous research.14 (link) Oligonucleotides specific for short hairpin RNAs (shRNAs) against circNR3C1 or BRD4 (Table S1 ) were inserted into lentiviral vector GV298 (Genechem, Shanghai, China). Cell lines were transfected using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer’s instructions. After selection for neomycin or puromycin (Invitrogen) resistance, stable cell lines were obtained.
4
Constructing Neuroblastoma Molecular Constructs
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Human circ‐CUX1 linear sequence (393 bp) was obtained from NB tissues by PCR (Appendix Table S2 ) and inserted into pLCDH‐ciR (Geenseed Biotech Co., Guangzhou, China). Human p200 CUX1 construct was provided by Dr. George Stratigopoulos, while p110 CUX1 was released by digestion or amplified using primers (Appendix Table S2 ), and subcloned into pcDNA3.1 (Invitrogen) or pCMV‐3Tag‐1A (Addgene, Cambridge, MA). Human EWSR1 cDNA (1,971 bp) and MAZ cDNA (1,482 bp) were provided by Dr. Ralf Janknecht or amplified from NB tissues with primers (Appendix Table S2 ), and their truncations were subcloned into pCMV‐N‐Myc or pCMV‐3Tag‐1A (Addgene). Mutation of circ‐CUX1 or EWSR1 was prepared with GeneTailor™ Site‐Directed Mutagenesis System (Invitrogen) and primers (Appendix Table S2 ). Oligonucleotides specific for shRNAs (Appendix Table S3 ) were inserted into GV298 (GeneChem Co., Ltd, Shanghai, China). Stable cells were screened by neomycin or puromycin (Invitrogen).
5
Engineered Transcription Factors and Vectors
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Human MZF1 coding sequence (CDS, 2205 bp), MZF1 cDNA (2920 bp), YY1 cDNA (1245 bp) and corresponding truncations were obtained from NB tissues by PCR primers (Table S2 ), and inserted into pcDNA3.1 (Invitrogen), pEGFP-N1, pCMV-3Tag-1C, pCMV-C-Flag, pCMV-N-MYC (Addgene, Cambridge, MA), or lentiviral expression vector CV186 (Genechem Co., Ltd, Shanghai, China), respectively. Human HK2 and PGK1 expression vectors were obtained from Genechem Co., Ltd. Mutation and frame-shift deletion of GFP or MZF1-uORF was prepared with GeneTailorTM Site-Directed Mutagenesis System (Invitrogen) and primers (Table S2 ). Oligonucleotides encoding short hairpin RNAs (shRNAs) specific for MZF1, HK2, PGK1, MZF1-uORF, or YY1 (Table S3 ) were subcloned into GV298 (Genechem Co., Ltd). Single guide RNAs (sgRNAs) were designed using Guide Design Resources (http://crispr.mit.edu ) to target upstream or downstream region relative to transcription start site of MZF1 (Table S2 ), and inserted into dCas9-VPR or dCas9-BFP-KRAB (Addgene), respectively. Stable cell lines were screened by administration of neomycin or puromycin (Invitrogen).
6
Modulation of circSTAU2 expression in cellular processes
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Oligonucleotides of miR-589 mimics, CAPZA1-short hairpin RNA (shRNA), MBNL1-siRNA, KHDRBS3-siRNA, and negative control (NC) were synthesized by GeneChem (Shanghai, China). The MBNL1 plasmid (GeneChem, Shanghai, China) was used for MBNL1 overexpression. A specific shRNA for circSTAU2 was inserted into GV298 (GeneChem, Shanghai, China) to knock down the expression of circSTAU2. Oligonucleotides encoding the circSTAU2 sequence were synthesized by TSINGKE (Wuhan, China) and cloned into pLCDHciR (GeneChem, Shanghai, China) for circSTAU2 overexpression. The sequences are available in Supplementary Table S1
7
Lentiviral Vector Construction for POU6F1 and RORA
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Human POU6F1 cDNA (1836 bp) and RORA cDNA (1671 bp) were obtained by PCR using the vector LV201-POU6F1 and LV205-RORA (Guangzhou FulenGen Co., Ltd.) and then inserted into CV186 lentivirus vector containing sequence which could express both red fluorescent protein and anti-puromycin protein (Genechem Co., Ltd., Shanghai, China). The POU6F1 PCR fragments and pCMV-HA were double-digested with EcoR I and Xho I and ligated to construct pCMV-HA-POU6F1. Meanwhile, the pCMV-3Tag-1-RORA vector was constructed with BamH I and Hind III. In addition, independent single guide RNAs (sgRNAs) targeting the downstream region of the POU6F1 transcription start site and oligonucleotides specific of shRNAs for RORA were inserted into dCas9-BFP-KRAB (Addgene) and GV298 vector (Shanghai GeneChem Co., Ltd), respectively. The primer sequences were listed in Supplementary Table 5 . Stable cell lines were constructed with puromycin for 3–4 weeks.
8
Cloning and Stable Expression of circREOS
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Linear circREOS (1811 bp) was synthesized by TSINGKE (Wuhan, China) and inserted into pLCDH-ciR (Geenseed Biotech Co., Guangzhou, China). Human MYC and HuR cDNA were purchased from Miao Ling Plasmid (Wuhan, China) and subcloned into pCMV-3Tag-1A. The insertion of Oligonucleotides (Supplementary Table S4 ) into GV298 (GeneChem Co., Ltd) was used for sh-circREOS construction. Stable cell lines treated with lentiviruses were obtained by selection with puromycin (2 μg/mL) for 2-3 weeks.
9
Generation of RBM15 Truncations and Stable Cell Lines
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Human RBM15 cDNA (2934 bp) was synthesized by TsingKe Biotech Company (Wuhan, China), and the truncations of RBM15 were obtained by PCR amplification with differential primer pairs (Table S2 ) and subcloned into pCMV‐3Tag‐1A (Addgene, Cambridge, USA). Oligonucleotides specific for shRNAs against circ‐CTNNB1 or RBM15 (Table S2 ) were inserted into GV298 (Genechem Co., Ltd., Shanghai, China). Stable cell lines were obtained, followed by selection with neomycin or puromycin (Invitrogen) for 2–3 weeks.
10
Molecular Cloning and Cell Line Generation
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Human HNF4A and MYCN expression vectors were provided by Dr. David Martinez Selva [16 (link)] and Dr. Arturo Sala [17 (link)], respectively. Human HNF4A-AS1 cDNA (648 bp), hnRNPU cDNA (2478 bp), CTCF cDNA (2184 bp), and their truncations were amplified from NB tissues (Additional file 1: Table S2 ) and subcloned into pcDNA3.1 (Invitrogen), pCMV-3Tag-1C, pCMV-N-Myc, and pGEX-6P-1 (Addgene, Cambridge, MA), respectively. Mutation of short hairpin RNA (shRNA)-targeting site of HNF4A-AS1 or hnRNPU RGG residues was performed with GeneTailorTM Site-Directed Mutagenesis System (Invitrogen) and primers (Additional file 1: Table S2 ). Oligonucleotides encoding shRNAs specific for HNF4A, HK2, SLC2A1, MYCN, HNF4A-AS1, hnRNPU, or CTCF (Additional file 1: Table S3 ) were subcloned into GV298 (Genechem Co., Ltd, Shanghai, China). Single guide RNAs (sgRNAs) targeting downstream region of HNF4A-AS1 transcription start site (Additional file 1: Table S3 ) were inserted into dCas9-BFP-KRAB (Addgene). Stable cell lines were screened by administration of neomycin or puromycin (Invitrogen).
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