WT and Hifi-Cas9 were purchased from IDT. The gRNAs used in this study were designed using Benchling (https://www.benchling.com/crispr ) and their sequences were shown in Additional file 2 : Table S1 [6 (link)]. The gRNAs were obtained either through in vitro transcribed by a MEGAshortscript™ T7 Transcription kit (ThermoFisher, Cat# AM1354) or ordered through IDT as Alt-R crRNAs or sgRNAs. For each electroporation, 50 pmol of Alt-R gRNA and 50 pmol of Cas9 were mixed and incubated at room temperature for 10 min to form ribonucleoprotein (RNP). Buffer R (from the Neon system kit) was added to the RNP to a final volume of 10 µl; 200,000 single cells were electroporated using a Neon system (ThermoFisher) with the setting of 1600 V, 10 ms width and 3 pulses. For the HDR study, 30 pmol ssODN was mixed with 50 pmol RNP before the electroporation. The cells were seeded in 1 well of a 24-well plate immediately after electroporation.
Neon system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Neon system is a compact, benchtop instrument designed for automated cell isolation and single-cell sample preparation. It utilizes microfluidic technology to capture and process individual cells from a heterogeneous sample, enabling downstream analysis such as single-cell genomic, transcriptomic, or proteomic studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (10 mentions)
Neon transfection kit, by Thermo Fisher Scientific (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Resuspension buffer, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Calphos kit, by Takara Bio (2 mentions)
Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Pierce gaussia luciferase glow assay kit, by Thermo Fisher Scientific (1 mentions)
Smartpool sirna, by Horizon Discovery (1 mentions)
Interferin, by Polyplus Transfection (1 mentions)
Rpmi 8226, by Leibniz Institute DSMZ (1 mentions)
Opti mem serum free media, by Thermo Fisher Scientific (1 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (1 mentions)
Fitc conjugated goat anti mouse secondary fab, by Jackson ImmunoResearch (1 mentions)
Metafluor software, by Molecular Devices (1 mentions)
81 protocols using neon system
1
CRISPR-Cas9 Electroporation Protocol
2
Quantifying NF-κB Pathway Activation
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Cells were transfected using the Neon system (Life Technologies) with plasmids expressing the firefly luciferase either driven either by a promotor containing 6 NF-κB binding sites (NF-κB-Luc) or by the control CMV promotor (CMV-Luc) and incubated in the presence of different concentrations of compounds. Next cells were harvested and analyzed for luciferase expression. Signal from NF-κB-Luc reporter was normalized according to the signal from the control CMV-Luc reporter.
3
Cell Culture Protocols for Cell Lines
4
CRISPR-based P2X4 and ALG2 Knockout
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The operation followed detailed instructions of the published protocol (Cong et al., 2013 (link)). In brief, 20-bp target single-guide RNA sequences were obtained by screening P2X4 mRNA sequence with CRISPR DESIGN online software (http://crispr.mit.edu/ ), and two sequences were chosen: 5′-AGTCCCGAGTGTCGAGGCGC-3′ (target 1) and 5′-GGGTGCTGTTATGGACGTGT-3′ (target 2). After being annealed with their reverse complementary sequences, the short double-strand DNAs were ligated with Bbs I–digested pX330-2A-GFP that was generously provided by L. Mei, and the new constructs were named pX330-2A-GFP/P2X4-1 and -2, respectively. For ALG-2, the chosen guide RNA sequences were 5′-GCTGCCTACTCCTACCGCCC-3′ (target 1) and 5′-TGTCTTCCGGACCTACGACA-3′ (target 2), the annealed DNAs were further ligated with Bbs I–digested pX330-2A-GFP, and the new constructs were named pX330-2A-GFP/ALG2-1 and -2, respectively. C2C12 cells were transfected with individual pX330-2A-GFP/P2X4 using the Neon system (Life Technologies) following optimized protocol and cultured for 72 h before use. The transfection efficiency for C2C12 cells was >90%.
5
Gaussia Luciferase Assay in GSCs
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Gaussia luciferase plasmid (Addgene #22522) was transfected in GSCs using the Neon system (Life Technologies). 8000 transfected cells were plated in a 96-well plate 24h later and treated as indicated. Supernatants were then collected and analyzed for Gaussia luciferase activity using the Pierce Gaussia Luciferase Glow assay kit following manufacturers’ protocol (Thermo Scientific, 16160).
6
Plasmid Transfection and Cav1 Silencing in MIN6 Cells
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Plasmid DNAs (2 μg) were transfected into 1 million MIN6 cells using the NEON system (Life Technologies) using two 1200 V pulses and a pulse width of 20 ms. Cav1 RNA interference was achieved by transfecting MIN6 cells with Dharmacon SMARTpool siRNA against Cav1 (Cat. L-058415-00-0005) using Lipofectamine RNAiMAX (Life technologies) according to the manufacturer's instructions achieving a final siRNA pool concentration of 100 nM. Dharmacon SMARTpool scrambled siRNA was used at 100 nM as a control. Cells were analyzed 48 h after transfection.
7
Analyzing Cell Cycle Regulators via RNAi
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siRNA oligos were synthesized by Dharmacon: CCNA2 (MU-003205-02-002), CCNE1 (MU-003213-02-0002), CCNE2 (MU-003214-02-0002), CDKN1A (MU-003471-00-0002), CCND1 (MU-003210-05-0002), RB1 (MU-003296-03-0002) or IDT: CCNB1 (hs.Ri.CCNB1.13.1), GMNN (hs.Ri.GMNN.13.1), CDT1 (hs.Ri.CDT1.13.2), MYC (hs.Ri.MYC.13.2), and Negative Control DsiRNA (51-01-14-04). The oligos were electroporated into MCF10A cells following manufacturer’s instruction (Neon system, Life Technologies). Cells were fixed for IF or lysed for western blotting 20 hours (for short-live proteins: Cyclin A2, Cyclin B1, Cyclin E, Cyclin D1, c-Myc, p21, Geminin, and Cdt1) or 48 hours (for longer-live proteins: Rb) after the electroporation.
8
Silencing DNA Repair Genes Using RNAi
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RNAi oligos against EXO1 (Cat. N. SI02665138—EXO1#1 and Cat. N. SI02665145—EXO1#2), RECQ1 (Cat. N. GS5965, SMARCAL1 (Cat. N. GS50485—equimolar mixture of 4 the siRNAs) and MRE11 (Cat. N. SI02665180) were from Qiagen. Oligos were used at the final concentration of 40, 10, 20 and 20 nM, respectively. The siRNA against DNA2 was a kind gift from Dr Vindigni (14 (link)). Transfection was performed using INTERFERin (Polyplus) according to the manufacturer's instructions. As a control, a siRNA duplex directed against GFP was used. DNA2 was silenced using the pLKO-shDNA2 plasmid (Addgene #31951). The plasmid was transfected by nucleofection using the Neon system (Life Technologies). Decrease in protein levels was confirmed by western blotting at 48 h after transfection.
9
Transfection of Human Multiple Myeloma and Bladder Cancer Cells
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FGFR3-negative (RPMI-8226) and wild-type (LP1) human MM cell lines were obtained from the German Collection of Microorganisms and Cell Cultures [DSMZ; Braunschweig, Germany]; FGFR3 mutant MM cells (KMS-11; Y373C) derived from a MM patient and established at Kawasaki Medical School [32] (link), were generously provided by Dr. P Leif Bergsagel. The mutant FGFR3 bladder cancer cell line, MGHU3 (Y375C), a kind gift from Dr. Margaret Knowles (University of Leeds, Leeds, UK), was derived from a grade 1 tumor [33] (link). MM and UC cells were maintained in RPMI 1640 (Hyclone; Thermo Scientific, Rockford, IL) and HeLa and HEK293 cells (ATCC) in DMEM (Hyclone), both media supplemented with 10% fetal bovine serum (Invitrogen). Transient transfection of HeLa and HEK293 cells was achieved using Lipofectamine 2000 (Life Technologies; Grand Island, NY) according to the manufacturer’s protocol and MM and UC transfected lines using the Neon system (Life Technologies). Following the manufacturer’s procedure, 1×106 UC or 2×106 MM cells were suspended in 100 µl suspension solution containing 5 µg siRNA (Dharmacon) or plasmid and pulsed under program 3 for UC and program 15 (KMS-11) or 20 (RPMI-8226) for MM cells.
10
Fibroblast and Cell Line Culture and Transfection
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Human fibroblasts were cultured in DMEM (Gibco) supplemented with 20% fetal calf serum (FCS) and 1% penicillin/streptomycin. bs mEFs and HeLa cells were cultured in a similar medium containing 10% FCS. Human fibroblasts and bs mEFs were maintained under hypoxic conditions (3% O2, 5% CO2) at 37°C. HeLa cells were maintained under normoxic conditions (5% CO2) at 37°C.
Transfection of human fibroblasts and HeLa cells was carried out using Lipofectamine 2000 (Life Technologies) according to manufacturer's instructions. Briefly, up to 0.5 µg plasmid DNA was combined with 1.5 µl Lipofectamine 2000 per transfection and added to cells in a total volume of 500 µl opti-MEM serum-free media (Gibco) for 4 h. Transfection mixes were then replaced with full media, and cells were allowed to recover for 18–24 h prior to fixation or imaging. Transduction of bs mEFs was carried out using the Neon system (Life Technologies) according to manufacturer's instructions. Briefly, 0.5 µg plasmid DNA was combined with cells in suspension in a total of 10 µl buffer ‘R’ per electroporation and loaded into a 10 µl electroporation tip. Cells were electroporated with a single pulse at 1350 V with a width of 30 ms and allowed to adhere and recover for 18–24 h in full media prior to fixation or imaging.
Transfection of human fibroblasts and HeLa cells was carried out using Lipofectamine 2000 (Life Technologies) according to manufacturer's instructions. Briefly, up to 0.5 µg plasmid DNA was combined with 1.5 µl Lipofectamine 2000 per transfection and added to cells in a total volume of 500 µl opti-MEM serum-free media (Gibco) for 4 h. Transfection mixes were then replaced with full media, and cells were allowed to recover for 18–24 h prior to fixation or imaging. Transduction of bs mEFs was carried out using the Neon system (Life Technologies) according to manufacturer's instructions. Briefly, 0.5 µg plasmid DNA was combined with cells in suspension in a total of 10 µl buffer ‘R’ per electroporation and loaded into a 10 µl electroporation tip. Cells were electroporated with a single pulse at 1350 V with a width of 30 ms and allowed to adhere and recover for 18–24 h in full media prior to fixation or imaging.
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