4d nucleofector x kit

Purified monocytes were left untreated or transfected with 300 nM control siRNA, siCA12, or siHIF1A using The P3 Primary Cell 4D-Nucleofector X Kit with Lonza 4D Nucleofector (Lonza). All siRNA duplexes were purchased from GenePharma, and their sequences are listed in Supplemental Table 5.

The preparation of PRIP-DKO MEFs was conducted as previously reported [2] (link), [21] (link). MEFs stored in liquid nitrogen were grown in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 15% fetal bovine serum (FBS; Gibco/Life Technologies, Carlsbad, CA) and 1% penicillin/streptomycin (Nakalai Tesque Inc., Kyoto, Japan). Cultures were maintained at 37°C in a humidified 5% CO₂ incubator, as previously described [2] (link). Plasmids were transfected into MEFs using 4D-Nucleofector and a 4D-Nucleofector X Kit (Lonza, Basel, Switzerland), according to the manufacturer's instructions. Briefly, cells (5×10⁵ cells) were resuspended in 100 µL of 4D-Nucleofector solution and transfected with 2 µg of each plasmid. The transfected cells were seeded onto glass coverslips and allowed to adhere overnight in culture medium (DMEM containing 15% FBS without antibiotics).

For EGFP disruption assay 200,000 U2OS.EGFP cells were nucleofected with 1 μg of px-Cas9 plasmids bearing a sgRNA designed to target EGFP using the 4D-Nucleofector™ X Kit (Lonza), DN100 program, according to the manufacturer’s protocol. After electroporation, cells were seeded in 96-well plates and transferred to 24-well plates after 48 h for growth expansion.
For editing analyses of genomic loci, 100,000 HEK293T cells were seeded in a 24-well plate 24 h before transfection. Cells were then transfected either with 1 μg of pX-Cas-sgRNA plasmid expressing both the Cas9s and the sgRNAs, or with 500 ng of a pX-Cas9 plasmids and 250 ng of a pUC-sgRNA constructs, using the TransIT-LT1 reagent (Mirus Bio) according to the manufacturer’s protocol. Cell pellets were collected 3 days post-transfection for indel evaluations. For base editing experiments, cells were co-transfected as described above with 750 ng of pCMV-ABE8e containing the specific Cas9s and 250 ng of pUC-sgRNA.

The differential expression profiles of microRNA between G-ADSCs and M-ADSCs were detected by microRNA microarray, and miR669p was extremely significantly increased after D-mannose treatment. For further exploration, ADSCs were transfected with the miR669b-5p inhibitor. Briefly, ADSCs were seeded at a density of 2 × 10⁵ cells per well in 6-well plates in culture medium without antibiotics. Cells were transfected with a mission synthetic microRNA inhibitor, mmu-miR-669b-5p, and negative control (Sigma, USA) using the 4D-Nucleofector™ X Kit (Lonza) at 70% confluence following the manufacturer's instructions. Transfection efficiency was determined by quantitative real-time PCR at day 1 and day 2 after transfection.
The transfected ADSCs were induced in adipogenic medium. The gene expressions of Pparg and Fabp4 were analyzed via real-time PCR analysis after adipogenic induction, and Oil Red O staining was applied after 14 days of adipogenic induction.

Cells were transfected using the SF cell line 4D-nucleofector X kit (Lonza) with the CN-114 program according to the manufacturer’s protocol. One million cells were transfected with a total of 2,000 ng of plasmid DNA (1,000 ng of each DdCBE monomer). Cells were split 24 h after transfection and used for experiments. See Supplementary Table 2 for a list of plasmids used for transfection.

Two sgRNAs targeting human CD55 exons were designed using the Broad Institute’s GPP sgRNA design portal and synthesized as chemically modified sgRNAs by Synthego. CD55-Cr1 is predicted to recognize a sequence in exon 1 of CD55 and has the sequence: GGGCCCCUACUCACCCCACA. CD55-Cr8 is predicted to recognize a sequence in exon two and has the sequence: CUGGGCAUUAGGUACAUCUG. Experiments using dual sgRNAs typically produce large deletions between the Cas9 binding sites as well as smaller indels, leading to frameshift mutations (Mandal et al., 2014 (link)). Ribonucleoprotein (RNP) complexes containing one or both sgRNAs were prepared by slowly adding 300 pmol of each sgRNA to 150 pmol Cas9 protein in a 10 µl final volume with nuclease-free water and incubating at room temperature for 10 min. On day 2 after thawing CD34 + cells, the RNP complexes were added to 1 × 10⁵ cells in 40 µl of P3 nucleofection buffer from the 4D-Nucleofector X kit (Lonza). Half of the mixture was loaded to each well of a 16-well nucleofection cassette and nucleofected using the using E0-100 program with the 4D-Nucleofector Lonza Amaxa. After nucleofection, cells were transferred to 6 ml fresh cPIMDM and incubated at 37°C in 5% CO2 in air.

Three hundred ten picomoles effector and 500 picomoles sgRNA were used to transfect 2 million cells using a 4D-Nucleofector X Kit (Lonza). Cells were recovered for 72 h at 37°C and genomic DNA was isolated using the NucleoSpin Blood L Midi Kit (Macharey Nagel). Nextera compatible primers with diversity stub sequences ranging between 1 and 5 nt were pooled to 500 pM to amplify 90 ng/μL gDNA in NEBnext Q5 Master mix (NEB) over 23 amplification cycles. Ten 100 μL polymerase chain reaction (PCR) reactions of each isolated gDNA condition were pooled and processed with 1 × SPRI beads (manufacturer) and sequenced using a 150 cycle MiSeq kit (Illumina). Sequences were processed using CRISPResso2, and indel profiles were visualized using SeqLogo.