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Lullaby transfection reagent

Manufactured by Ozyme
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Lullaby is a transfection reagent designed to facilitate the introduction of genetic material into cells. It is a non-viral delivery system that enables the efficient and gentle delivery of DNA, RNA, or other molecules into a variety of cell types. The core function of Lullaby is to effectively transport the desired genetic material across the cell membrane and into the target cells, allowing for gene expression or gene silencing studies.

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Lab products found in correlation

Opti mem, by Thermo Fisher Scientific (2 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Transit lt1 transfection reagent, by Mirus Bio (1 mentions) Sigenome smartpool sirna, by Horizon Discovery (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Aphidicolin, by Merck Group (1 mentions) Sirnas, by Horizon Discovery (1 mentions) Topotecan, by Merck Group (1 mentions) Formaldehyde, by Thermo Fisher Scientific (1 mentions) Biomek fx liquid handling robot, by Beckman Coulter (1 mentions) Acumen explorer ex3 laser scanning microplate cytometer, by SPT Labtech (1 mentions) Reversine, by Merck Group (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Quantitect primer kits, by Qiagen (1 mentions) Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Lullaby reagent (11 citations) Lullaby (9 citations) Lullaby siRNA transfection reagent (3 citations)

8 protocols using lullaby transfection reagent

1

Transient RPC5 knockdown and overexpression in HEK293T cells

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HEK293T cells (a kind gift from Dr Sebastien Guettler) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2. For transient knockdown, 20 nM siRNA of either ONTARGETplus siRNA for RPC5 (Horizon Discovery) or AllStars negative control siRNA (Qiagen) was used per well. This was transfected using Lullaby transfection reagent (Oz Biosciences) as per the manufacturers’ instructions. The sequences for each RPC5 siRNA are as follows: 5′-UGGAUAAGGCUGACGCCAA-3′, 5′-GGGAGCAGAUUGCGCUGAA-3′, 5′-CGACGAGACCAGCACGUAU-3′, 5′-CCUCGAUGACCUACGAUGA-3′. For transient overexpression of RPC5 (HA-tagged full-length ΔtWHD2 or ΔC), DNA (1.5 μg) was transfected in using Fugene HD transfection reagent (Promega) as per the manufacturers’ instructions.
Ramsay E.P., Abascal-Palacios G., Daiß J.L., King H., Gouge J., Pilsl M., Beuron F., Morris E., Gunkel P., Engel C, & Vannini A. (2020). Structure of human RNA polymerase III. Nature Communications, 11, 6409.
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2

Screening Ubiquitination Machinery in TMPRSS2-HiBiT Cells

Cited 1 time
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TMPRSS2-HiBiT cells were screened with an siRNA library targeting Ubiquitination-related machinery47 (link). Briefly, 25 ng of siRNA was mixed with Lullaby transfection reagent (OzBiosciences) and diluted in Opti-MEM media. The transfection mixture incubated at room temperature for 20 min, and was added to 20 μL of HITES + 10%FBS media with 2000 cells. Following 72 h knockdown, Lytic HiBiT luciferase assays were performed using manufacturer’s protocol. For conformational specific gene silencing, small interfering RNAs were selected and purchased from IDT, and transfected in cells using Lullaby siRNA transfection reagent, with Negative Control DsiRNA transfected as control. Subsequent analysis was performed after 72 h of knockdown.
Chen Y., Lear T.B., Evankovich J.W., Larsen M.B., Lin B., Alfaras I., Kennerdell J.R., Salminen L., Camarco D.P., Lockwood K.C., Tuncer F., Liu J., Myerburg M.M., McDyer J.F., Liu Y., Finkel T, & Chen B.B. (2021). A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry. Nature Communications, 12, 3907.
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3

Generating Stable Cell Lines and Silencing Genes

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Plasmid transfections to generate stable cell lines were performed with TransIT®-LT1 transfection reagent (Mirus). 2 × 105 cells per well were seeded in 6-well plates the day before transfection, transfection complexes containing 3 μl of the transfection reagent per μg of transfected DNA were prepared in Opti-MEM™ (Gibco) medium, incubated 20 min at room temperature and then added onto the cells. The medium was exchanged on the following day and antibiotic selection started on the third day after transfection.
For siRNA transfections, 2 × 105 cells per well were seeded into a 6-well plate and transfections were performed using the Lullaby transfection reagent (OZ Biosciences). Transfection complexes were prepared in Opti-MEM™ (Gibco) medium and consisted of 5.6 μl of the transfection reagent and 22 nM siRNA in a final volume 2 ml/well. Cells were transfected twice with the siRNAs, on the first and third days after seeding, and harvested one day after the second transfection.
Nasif S., Colombo M., Uldry A.C., Schröder M.S., de Brot S, & Mühlemann O. (2023). Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells. NAR Cancer, 5(3), zcad048.
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4

Downregulation of Human ATF-4 using siRNA

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The siRNA used to downregulate human ATF-4 and the non-targeting siRNA control were purchased from Dharmacon (siGENOME SMART pool siRNA). Cells were transfected with siRNA using Lullaby transfection reagent (OZ Biosciences) following the manufacturer’s instructions.
Tajan M., Hennequart M., Cheung E.C., Zani F., Hock A.K., Legrave N., Maddocks O.D., Ridgway R.A., Athineos D., Suárez-Bonnet A., Ludwig R.L., Novellasdemunt L., Angelis N., Li V.S., Vlachogiannis G., Valeri N., Mainolfi N., Suri V., Friedman A., Manfredi M., Blyth K., Sansom O.J, & Vousden K.H. (2021). Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy. Nature Communications, 12, 366.
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5

Effective siRNA Transfection for Protein Knockdown

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The transfection mix added to the cells (grown to 40–60% confluency) consisted of 1:10 (v/v) of the culture medium and contained 25 nM siRNA (ABCE1 siRNA, 5′-GAG GAG AGU UGC AGA GAU UU dTdT-3′ or Negative Control siRNA, 5′-AGG UAG UGU AAU CGC CUU G dTdT-3′, Microsynth) and 0.25% (v/v) Lullaby transfection reagent (OZBiosciences) dissolved in Opti-MEM (Thermofisher). Before addition to the cells, the mix was incubated at RT for 20 min to allow complex formation. After 24 h of incubation, the cells were split to a higher format including a PBS pH 7.4 washing step. On the next day, the transfection was repeated under the same conditions and 24 h later cells were harvested and immediately processed to yield translation-competent lysates.
Karousis E.D., Gurzeler L.A., Annibaldis G., Dreos R, & Mühlemann O. (2020). Human NMD ensues independently of stable ribosome stalling. Nature Communications, 11, 4134.
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6

Ionizing Irradiation Cellular Response

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Cells were cultured in DMEM (Invitrogen) supplemented with penicillin/streptomycin (Invitrogen), and 10% FCS (Invitrogen). Cells were incubated at 37 °C with 5% CO2 and 3% O2. Ionizing irradiation (IR) experiments were performed using a Cs137 Gamma Irradiator at the indicated doses followed by a recovery period of 30 min. Aphidicolin, MMS, NCS, HU and topotecan were purchased from Sigma. siRNAs were purchased from Dharmacon and transfected using the Lullaby transfection reagent as directed by the manufacturer (Oz Biosciences). Cells were analyzed 48–72 h after transfection.
Schmidt L., Wiedner M., Velimezi G., Prochazkova J., Owusu M., Bauer S, & Loizou J.I. (2014). ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress. DNA Repair, 24, 122-130.
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7

High-Throughput Screening of siRNA Impacts

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The primary screen was performed using the human siRNA Smart-pool siGenome library from Dharmacon, whereas the secondary screen was performed using the corresponding deconvoluted siRNA duplexes (where four siRNAs included in the pool were tested independently). Contact-inhibited RPE1 cells were reverse-transfected in triplicate 384-well plates using Lullaby transfection reagent [OZ Biosciences, 800 cells/well, 0.2 μL Lullaby/well, 18.75 nmol/L siRNA, 250 nmol/L reversine (Sigma)] using a Biomek FX liquid handling robot (Beckman). Cells were cultured for 96 hours and fixed with 4% formaldehyde (ThermoFisher, no. 28908), except for the deconvolution experiment, where cells were pulsed for 1 hour with 10 μmol/L EdU before fixation to label cells in S phase. Cells were subsequently permeabilized and blocked in PBS + 3% BSA + 0.2% Triton-X100 for 30 minutes and processed for immunofluorescence (IF) and EdU detection (LifeTechnology) according to the manufacturer’s protocol. Images were acquired and analyzed using an ArrayScan VTi-automated microscope (Cellomics), except for total cell counts, which were acquired on an Acumen Explorer eX3 laser scanning microplate cytometer (TTPLabtech). For each parameter, a median Z-score was derived using plate normalization [(well value – plate median)/plate Median Absolute Deviation].
Sansregret L., Patterson J.O., Dewhurst S., López-García C., Koch A., McGranahan N., Chao W.C., Barry D.J., Rowan A., Instrell R., Horswell S., Way M., Howell M., Singleton M.R., Medema R.H., Nurse P., Petronczki M, & Swanton C. (2017). APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability. Cancer discovery, 7(2), 218-233.
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8

Quantifying Gene Expression Knockdown

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Cells were transfected with scrambled or gene-targeting siRNA using Lullaby transfection reagent (Oz Biosciences). Cells were transfected twice with 25 nM siRNA, once every 48 h, then assayed 48 h after the second transfection.
To assess gene knockdown, RNA was extracted from cells using an RNeasy mini kit (Qiagen) and cDNA synthesized from 0.5 μg samples using a SuperScript III reverse transcriptase kit (Life Technologies) as specified by the manufacturer. qPCR was performed using PerfeCTa SYBR green FastMix (Quanta), with cDNA analysed in triplicate using a 7500 fast real-time PCR system (Applied Biosciences); data were extracted using Applied Biosystems 7500 software version 2.0. QuantiTect primer kits (Qiagen) were used to assess gene expression of GAPDH, PPAP2A/LPP1, PPAP2B/LPP3 and PPAP2C/LPP2, with all gene expression normalized against GAPDH as an internal control using the comparative Ct method (Schmittgen and Livak, 2008 (link)).
Susanto O., Koh Y.W., Morrice N., Tumanov S., Thomason P.A., Nielson M., Tweedy L., Muinonen-Martin A.J., Kamphorst J.J., Mackay G.M, & Insall R.H. (2017). LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells. Journal of Cell Science, 130(20), 3455-3466.
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