The lentiviral vector pLVX-EF1alpha-IRES-Puro-2xStrep-GFP or pLVX-EF1alpha-IRES-Puro-2xStrep-Orf9c (Addgene plasmid #141395 and #141393) was transfected into the HEK293T cells with packaging plasmids psPAX2 and pMD2.G using X-treme GENE nine transfection reagent (Roche). After 48 h, viral supernatant was collected and cellular debris was removed by syringe filtering (0.45 μm pore size; Millipore). H9 hESCs or S3 hiPSCs were incubated with virus supernatant (20% vol:vol) for 4 h and same infection was repeated after 24 h. Puromycin was added to select puromycin-resistant H9 hESCs or S3 hiPSCs clones after 48 h of viral infection.
Syringe filtering
Manufactured by Merck Group
Syringe filtering is a laboratory device used to filter small volumes of liquid samples. It consists of a syringe and a filter membrane, allowing for the removal of particulates from the sample prior to analysis or further processing.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (4 mentions)
X tremegene 9 transfection reagent, by Roche (4 mentions)
Ridascreen verotoxin elisa kit, by R-Biopharm (2 mentions)
X treme gene nine transfection reagent, by Roche (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Dreamtaq green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Primestar dna polymerase kit, by Takara Bio (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
7 protocols using syringe filtering
1
Lentiviral Transduction of Pluripotent Stem Cells
2
Induction and Quantification of Shiga Toxin
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3 ml LB was inoculated directly from glycerol stocks and grown overnight at 37 °C. 6 ml LB was inoculated 1/100 from overnight cultures and grown to an OD600nm = 0.6–0.8 (t = 0). Cultures were split 1:1 and phage lysis was induced in one half by the addition of Mitomycin C (MMC, 2 μg/ml). Subsequent growth/lysis in induced and un-induced cultures was monitored spectrophotometrically at OD600nm. For Stx toxin ELISA assays cultures were grown as above and growth/lysis allowed to proceed for 24 h. After 24 h, 1 ml culture was taken and live cells and cell debris removed by centrifugation (13,000 r.p.m, room temperature (RT)). Stx toxin containing supernatants were further sterilized by syringe filtering (0.22 μm; Milipore). The level of Stx toxin in each sample was assayed using the RIDASCREEN Verotoxin ELISA kit (R-Biopharm) according to manufacturer guidelines.
3
Lentiviral Transduction of Human iPSCs
4
Lentiviral Transduction of H9 Cells
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The lentiviral vector lentiCRISPRv2-puro was transfected into the HEK293T cells (ATCC) along with packaging plasmids including psPAX2 and pMD2.G (from Dr. Guang Hu in NIH) using the X-treme GENE 9 transfection reagent (Roche). Viral supernatant was collected, and cellular debris was removed by syringe filtering (0.45 μm pore size; Millipore). Human H9 cells cultured in mTesR medium were incubated with virus media for 4 h, followed with fresh mTesR medium culture for overnight. The same infection was repeated after 24 h. Puromycin was added to select puromycin-resistant H9 clones after virus infection for 48 h.
5
Stx Toxin Production Assay
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In total, 3 ml LB was inoculated directly from glycerol stocks and grown overnight at 37 °C. 6 ml LB was inoculated 1/100 from overnight cultures and grown to an OD600 nm = 0.6–0.8. Mitomycin C (2 µg ml−1) was added and lysis allowed to proceed for 24 h. After 24 h, 1 ml culture was taken and live cells and cell debris removed by centrifugation (13000 r.p.m.). Stx toxin containing supernatants were further sterilized by syringe filtering (0.22 µm; Milipore). The level of Stx toxin in each sample was assayed using the RIDASCREEN Verotoxin ELISA kit (R-Biopharm) according to manufacturer guidelines. Differences Stx2 production was assessed by ordinary one-way ANOVA with multiple comparisons where each strain was compared with Z1723.
6
Lentiviral Transduction of Human Pluripotent Stem Cells
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The lentiviral constructs including pLKO.1-TRC and lentiCRISPRv2-puro vectors were transfected into the HEK293T cells (ATCC) along with lentiviral packaging plasmids including psPAX2 and pMD2.G using the X-tremeGENE 9 transfection reagent (Roche), following the manufacturer's instructions, in Opti-MEM (Life Technologies) and incubated for 3-4 h at 37°C. After incubation for 24-48 h, the viral supernatant was collected and cellular debris was removed by syringe filtering (0.22 μm pore size; Millipore). For lentiviral transduction, lentivirus was added twice, 24 h after cell seeding and again after an additional 24 h. For every infection, S3 hiPSCs or H9 hESCs were infected by viruses overnight. Puromycin (1.0 μg/ml) treatment was used for selection of transduced PSCs after 3 days of virus infection and maintained throughout culture.
7
Generating ARID1A Knockout hESC Clones
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The lentiviral vector lentiCRISPRv2-puro was transfected into the HEK293T cells (ATCC) along with packaging plasmids including psPAX2 and pMD2.G (from Dr. Guang Hu in NIH) using the X-treme GENE 9 transfection reagent (Roche). Viral supernatant was collected and cellular debris was removed by syringe filtering (0.45 μm pore size; Millipore). Human H9 cells cultured in mTesR medium were incubated with virus media for 4h, followed with fresh mTesR medium culture for overnight. Same infection was repeated after 24 hr. Puromycin was added to select puromycin-resistant H9 clones after virus infection for 48h.
PCR screening of ARID1A -/-clones HESC clones were picked and expanded in mTesR medium. Genomic DNAs from each hESCs clone were purified by DNeasy Blood & Tissue Kits (Qiagen). PCRs with different primer sets were conducted by using DreamTaq Green PCR Master Mix (Thermo Scientific) or PrimeSTAR DNA Polymerase kit (Takara). PCR products were run on agarose gel to visualize the fragment size. For all primers, please see the Table S4.
PCR screening of ARID1A -/-clones HESC clones were picked and expanded in mTesR medium. Genomic DNAs from each hESCs clone were purified by DNeasy Blood & Tissue Kits (Qiagen). PCRs with different primer sets were conducted by using DreamTaq Green PCR Master Mix (Thermo Scientific) or PrimeSTAR DNA Polymerase kit (Takara). PCR products were run on agarose gel to visualize the fragment size. For all primers, please see the Table S4.
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