The Alt-R CRISPR RNA (crRNA):Alt-R trans-activating crRNA (tracrRNA) duplexes for targeting BCL-xL or BCL-2 were prepared by mixing 0.5 nmol crRNA and 0.5 nmol tracrRNA in 25 μL of Nuclease-Free Duplex Buffer (Integrated DNA Technologies, IDT) by incubation at 95 °C for 5 min and cooling to RT. Single-stranded ultramer DNA oligonucleotides (ssODN) (IDT) were used as the homology directed repair (HDR) donor templates. The sequences of gRNAs and ssODN templates are listed in Supplementary Table 2 . Ribonucleoprotein (RNP) complexes with recombinant Streptococcus pyogenes Cas9 protein (IDT) and crRNA:tracrRNA duplex were assembled by incubating 100 pmol of Cas9 and 120 pmol of gRNA for 10–20 min at room temperature. HeLa (2 × 105) cells were resuspended in 10 μL of NeonTM transfection system buffer R from the NeonTM transfection system 10 mL kit (Thermo Fisher), and the RNP complex along with 100 pmol ssODN template were electroporated into HeLa cells with the NeonTM transfection system (Thermo Fisher). After electroporation, HeLa cells were transferred to a 12-well plate for culturing. Edited pooled cells were analyzed for HiBiT insertion by assaying for luminescence using Nano-Glo® HiBiT lytic detection system (Cat #N3040, Promega) in a Biotek’s Synergy Neo2 multimode plate reader (Biotek) 48–72 h after electroporation and single clones were selected by serial dilution.
Neontm transfection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, United Kingdom
The Neon™ Transfection System is a laboratory equipment designed for efficient and reliable transfection of various cell types. It utilizes an electroporation-based method to introduce nucleic acids, such as plasmid DNA or RNA, into cells. The system provides a streamlined workflow and consistent results, making it a valuable tool for researchers in the life sciences.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Dual luciferase assay system, by Promega (3 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
L glutamine, by Merck Group (2 mentions)
L glutamin, by Euroclone (2 mentions)
On targetplus smartpool, by Horizon Discovery (2 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Nano glo hibit lytic detection system, by Promega (1 mentions)
Nuclease free duplex buffer, by Integrated DNA Technologies (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Jetpei dna transfection reagent, by Polyplus Transfection (1 mentions)
Metafectene reagent, by Biontex (1 mentions)
Human serum, by Merck Group (1 mentions)
Accumax, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Mission sic 001, by Merck Group (1 mentions)
Pe cy7, by BD (1 mentions)
Facs lsr 2, by BD (1 mentions)
49 protocols using neontm transfection system
1
CRISPR-Cas9 Knockout and HDR of BCL-xL/BCL-2 in HeLa cells
2
Astrocyte Crystallin Transfection and Glucose Modulation
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Transfection of astrocytes with βA1- and βA3-crystallin vectors was performed using NeonTM Transfection system according to manufacturer’s protocol (Thermo Fisher, USA; Cat# MPK10096). For appropriate transfection, cells were cultured for 48 h in medium containing 5 mM d -Glucose and then the medium was replaced with high glucose (30 mM) containing medium for another 6 h. WT astrocytes were infected with either GIPZ Cryba1 shRNA or PTPN1 (Ad-CMV-mNeonGreen-mPtpn1) overexpression construct for 48 h, and then high glucose medium was added to the cells for the next 6 h. For PTP1B inhibition, MSI-1436 (MedChemExpress, USA; Cat# HY-12219A) was added to βA1 KD astrocytes in culture at a dose of 10 μM24 (link), 1 h prior to addition of high glucose.
3
Generating SW480 PIWIL2-expressing cell lines
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SW480 was cultured in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Brazil),
at 37 °C in a humidified atmosphere of 95% air with 5% CO2. The stable SW480-PIWIL2 expressing (SW480-P) and SW480-empty vector (SW480-control)
cell lines were generated by transfecting SW480 cell line using PCDNA3/PIWIL2 or PCDNA3/empty plasmid via electroporation with a NeonTM Transfection System (Thermo Fisher Scientific, USA)
(supplementary Fig. 1 ). After 12h, infected cells were selected by G418 (500µg.mL-1), Sigma-Aldrich,USA) for 28 days. Survived cells were expanded for further analysis.
at 37 °C in a humidified atmosphere of 95% air with 5% CO2. The stable SW480-PIWIL2 expressing (SW480-P) and SW480-empty vector (SW480-control)
cell lines were generated by transfecting SW480 cell line using PCDNA3/PIWIL2 or PCDNA3/empty plasmid via electroporation with a NeonTM Transfection System (Thermo Fisher Scientific, USA)
(
4
CRISPR-Mediated KDM2B Knockout
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In order to knock out KDM2B gene, guide RNAs (gRNA) targeting exon 7 of KDM2B were designed and cloned into pX330 vector. 10 μg pX330 constructs containing gRNA were electroporated into Fy-hES-3 cells using NeonTM transfection system (MPK10096, Thermofisher). Two days later, the top 1% GFP positive cells were sorted by FACS and picked manually into matrigel-coated 96-well-plate at density of single cell per well and cultured in mTeSR1 medium containing 10 μM ROCK inhibitor. After 3 days, the medium was changed to fresh mTeSR1 medium and one week later until passage. Between 12 to 15 days, the surviving clones were passaged into 24-well plates and half of the cells were harvested for genotyping. The targeted deletion of exon 7 loci in KDM2B was assessed by Sanger sequencing. The losses of targeted deletion in KDM2B KO cell lines were further validated by Western blot and immunostaining. Detailed oligonucleotides used are listed in Table S1 .
5
siRNA Knockdown of SMPD1 in MDCK Cells
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siRNA negative controls and siRNAs for SMPD1 were purchased from Qiagen (Tokyo, Japan). MDCK cells (5 × 106 cells) were incubated with 500 pmol siRNA and then transfected using a NeonTM transfection system (Thermo Fisher Sci, Tokyo, Japan) [22 (link)]. After electroporation, cells were seeded to 24-well plates and incubated at 37 °C. Experiments were performed at 48 h after siRNA transfection [22 (link)].
6
Mammalian Cell Culture Protocols
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HEK293T and mammary cells were cultured at 37°C in DMEM containing 1 µg/ml of streptomycin, 100 units/ml of penicillin, and 10% fetal bovine serum (FBS). For treatment with estradiol, cells were grown in the same medium containing phenol red-free DMEM supplemented with 5% charcoal-filtered FBS (i.e., estradiol-stripped medium). All media were obtained from Hyclone Laboratories (USA). Plasmids were transfected to cells by using Lipofectamine with PLUS reagent (Thermo Fisher Scientific), NeonTM Transfection System (Thermo Fisher Scientific), Metafectene reagent (Biontex Laboratories, Germany), or jetPEI™ DNA Transfection Reagent (Polyplus-transfection, France).
7
Generation of Helq Knockout Mice
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Helq−/− mice were generated by CRISPR/Cas9 technology. Briefly, one single-guide RNA (sgRNA) were designed targeting the first exon of Helq and cloned into pSpCas9(BB)-2A-GFP (PX458) vector as previously described107 (link). The designed plasmid was electroporated into mouse BVSC ESCs using NeonTM Transfection System (Thermofisher) according to the manufacturer’s instructions. Two days later, GFP-positive cells were sorted by FACS and transferred manually into Matrigel-coated 96-well-plates with one single cell per well. Genomic DNA was extracted from the harvested cells and used for Sanger Sequencing. The targeted ESCs with 8-nt deletion in the two alleles of Helq were injected into blastocysts to generate chimeric mice. The chimeric mice were self-crossed to generate Helq−/− mice. Genomic DNA was extracted to confirm the genotype.
8
Transfection of CD37-WT-psGFP2-N1 in CD37KO BJAB Cells
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CD37-WT-psGFP2-N1 (2 µg/mL) constructs were transfected into CD37KO BJAB lymphoma cells using the NeonTM transfection system (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells were resuspended in RPMI1640 with 10% heat-inactivated foetal bovine serum (FBS) and 1% ultra-glutamine (UG) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and maintained at 37 °C with 5% CO2 for 24 h after transfection.
9
Electroporation of Human Islet Cells
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Human islets were dispersed into a single cell suspension by enzymatic digestion (Accumax, Invitrogen). For each electroporation pulse with 10 μl tips of the NeonTM transfection system (Thermo Fisher Scientific), 150,000 cells were resuspended in 5 μl buffer R. Following CRISPR/Cas9 RNP assembly, CRISPR/Cas9 RNP complexes were added to cells in buffer R. Electroporation conditions tested in Figure S1 A consisted of: V1 = 1100V, 1 pulse, 40 ms, V2 = 1500 V, 3 pulses, 10 ms, V3 = 1600 V, 3 pulses, 10 ms. V3 was used for following experiments. Immediately after the pulse, cells were transferred into 24 well plates in culture medium comprised of RPMI 1640 (Gibco), 2.25 g/dL glucose, 1% penicillin/streptomycin (v/v, Gibco), and 2% human serum (Sigma-Aldrich). Electroporated islet cells were cultured in an Orbi-Shaker (34–206, Genesee Scientific) until day 5 or 6 prior to further molecular or physiological analysis. One day following electroporation, GFP expression was evaluated.
10
Modulating SETDB2 and WWOX in Gastric Cancer
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MKN74 and MKN45 cells with high SETDB2 expression were transfected with SETDB2 siRNAs (final concentration, 50nmol; Hs_SETDB2_0144, Sigma-Aldrich Japan, Ishikari, Japan) or negative control siRNA (Mission_SIC-001, Sigma-Aldrich Japan) using electroporation (NeonTM transfection System, Thermo Fisher Scientific Inc.). AGS and NUGC3 cells with low SETDB2 expression were transfected with the SETDB2/pcDNA3 expression vector (1 μg) or with the empty pcDNA3 vector using the FuGENE HD Transfection Reagent (Promega Corporation, Madison, WI). Transfection of WWOX siRNA (Hs_WWOX_9365, Sigma-Aldrich Japan) was conducted in GC cells by electroporation. Transfected cells were harvested after 48 or 72 hours of culture, and total RNA and protein was then extracted.
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