huES6-derived NPCs were differentiated to hCA3 for 14DIV. Differentiating hCA3 population were dissociated into a single cell suspension by enzymatic dissociation by treating with Accutase (Stem cell technologies). Cells were collected in PBS containing 0.1%BSA on ice. The cell suspension obtained was filtered with 22-μm filter (Millipore) and kept in cold PBS-BSA solution. Before counting, trypan blue was added to the cell suspension and the viability, cell density and presence of clumps were assayed on a TC20 Automated Cell Counter. Cell suspensions at a final dilution of 2,500 cells/μL and containing no less than 85% live cells were taken for the encapsulation and later steps. All reagents for encapsulation, cDNA synthesis, library preparation and sequencing were from Illumina Inc. Cell suspensions were loaded into a ddSEQ cartridge in a ratio of 21.5 μL Cell enzyme mix to every 4.5 μL cell suspension. 60 ul of 3′ barcode suspension mix and encapsulation oil were also loaded on the cartridge. The chip was then processed on a ddSEQ Single-Cell Isolator for about 5 minutes to generate single cell droplets.
2.2 μm filter
Manufactured by Merck Group
The 2.2 μm filter is a laboratory filtration product designed for the separation and isolation of particles, cells, and other materials from liquids. It features a pore size of 2.2 micrometers, which allows for the effective filtration of a wide range of samples. The filter's core function is to provide a reliable and consistent method for sample preparation and purification in various research and analytical applications.
Automatically generated - may contain errors
3 protocols using 2.2 μm filter
1
Single-cell RNA-seq of hCA3 Neurons
2
Lentiviral Expression of Mutant PP2Ac
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pLVSIN nFLAG3 human PP2Ac, PME-1, and PP6c were previously described (7 , 43 (link)). pLVSIN nFLAG3 human PP2Ac S24A, S30A, T40A, S43A, T78A, S93A, T219A, S225A/T227A, T235A, S285A, and T301A, pcDNA3 nFLAG2 PP2Ac and PP4c, pLVmCherry for non-target shRNA (shNT) and PME-1 targeting shRNA (shPME-1) were generated using InFusion HD Cloning Kit (Takara Bio). The sequences of shRNA are as follows: shNT, 5′-CAACAAGATGAGAGCACCA-3′, shPME-1#1, 5′-GGGCGATACATCTGAGTTCA-3′.
To produce lentiviruses, pLVSIN, a packaging plasmid (psPAX2), and a coat protein plasmid expressing vesicular stomatitis virus G protein (pMD2.G) were diluted in Opti-MEM containing Polyethylenimine MAX (PEI, Polysciences). The mixture was added to Lenti-X 293T cells (Takara Bio) Medium was replaced with fresh medium after 8 h, and cells were cultured for 48 h. The viral supernatants were filtered using a 2.2 μm filter (Millipore) and added to cells.
To produce lentiviruses, pLVSIN, a packaging plasmid (psPAX2), and a coat protein plasmid expressing vesicular stomatitis virus G protein (pMD2.G) were diluted in Opti-MEM containing Polyethylenimine MAX (PEI, Polysciences). The mixture was added to Lenti-X 293T cells (Takara Bio) Medium was replaced with fresh medium after 8 h, and cells were cultured for 48 h. The viral supernatants were filtered using a 2.2 μm filter (Millipore) and added to cells.
3
Lentiviral Transduction Protocols for shRNA and Overexpression
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pLVmCherry for shNT and shSET were previously generated (4 (link)). The sequences of shRNA are as follows: shNT, 5′-CAACAAGATGAGAGCACCA-3′, shSET#1, 5′-GGATGAAGGTGAAGAAGAT-3′, and shSET#2 5′-GATGAAGAGGCACTGCATT-3′. shRNA complementary DNA strands for Bmi-1 (shBmi-1: 5′-AGAGTTCGACCTACTTGTA-3′) with flanking sequences were annealed and ligated into the MluI–ClaI sites of pLVmCherry. DOX-inducible shRNA-expressing pTRIPZ plasmids were purchased from Horizon (control shRNA: RHS4743; shSET: RHS4696-201903403 and RHS4696-201904577). pLVSIN nFLAG3 human Bmi-1 and pLVSIN cFLAG3 human myr-Akt were prepared by amplifying DNA of the target gene by PCR and incorporating it into a vector using the InFusion HD Cloning Kit (Takara Bio).
To produce lentiviruses, pLV and pTRIPZ plasmids, a packaging plasmid (psPAX2), and a coat protein plasmid expressing vesicular stomatitis virus G protein (pMD2.G) were diluted in 333 μl of Opti-MEM, containing 2.5 μl of Polyethylenimine MAX (Polysciences). The mixture was added to Lenti-X 293T cells (Takara Bio) cultured in 6-well plates, incubated for 8 h, and replaced with 1.5 ml of medium. After 48 h, viral supernatants were filtered using a 2.2 μm filter (Millipore) and added to cells.
To produce lentiviruses, pLV and pTRIPZ plasmids, a packaging plasmid (psPAX2), and a coat protein plasmid expressing vesicular stomatitis virus G protein (pMD2.G) were diluted in 333 μl of Opti-MEM, containing 2.5 μl of Polyethylenimine MAX (Polysciences). The mixture was added to Lenti-X 293T cells (Takara Bio) cultured in 6-well plates, incubated for 8 h, and replaced with 1.5 ml of medium. After 48 h, viral supernatants were filtered using a 2.2 μm filter (Millipore) and added to cells.
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