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Sf016 200

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The SF016-200 is a laboratory equipment product. It is designed for general laboratory use. The core function of the SF016-200 is to provide a controlled environment for various scientific experiments and procedures.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions) Doxycycline, by Merck Group (2 mentions) Chir99021, by Bio-Techne (2 mentions) β mercaptoethanol, by Merck Group (2 mentions) Cgp77675, by Bio-Techne (2 mentions) G 6976, by Bio-Techne (2 mentions) Turbofect transfection reagent, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Sodium pyruvate, by Merck Group (1 mentions) L glutamine, by Merck Group (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions)

7 protocols using sf016 200

1

Maintaining Mouse Embryonic Stem Cells

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Mouse embryonic stem cells (mESCs) R1 were maintained in Dulbecco’s Modified Eagle Medium (DMEM, BI, 01-052-1ACS) high glucose media containing 10% fetal bovine serum (FBS, Gibco, 10099141), 10% knockout serum replacement (KSR, Gibco, 10828028), 1 mM sodium pyruvate (Sigma, S8636), 2 mM L-Glutamine (Sigma, G7513), 1,000 U/ml leukemia inhibitory factor (LIF, Millipore, ESG1107) and penicillin/streptomycin (Gibco, 15140-122) at 37°C with 5% CO2.
The 2i culture condition was used as described previously (Chappell et al., 2013 (link)). The commercial ESGRO-2i Medium (Merck-Millipore, SF-016-200) was also used when necessary. We found that in 2i medium, Adnp-/- ESCs adopted morphology indistinguishable to that of control ESCs, and maintain self-renewal capacity for more than 20 passages that we tested.
Sun X., Yu W., Li L, & Sun Y. (2020). ADNP Controls Gene Expression Through Local Chromatin Architecture by Association With BRG1 and CHD4. Frontiers in Cell and Developmental Biology, 8, 553.
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2

Maintenance of mouse embryonic stem cells

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Mouse embryonic stem cells (mESCs) R1 were maintained in Dulbecco’s Modified Eagle Medium (DMEM, BI, 01-052-1ACS) high glucose media containing 10% FBS (Gibco, 10099141), 10% knockout serum replacement (KSR, Gibco, 10828028), 1 mM sodium pyruvate (Sigma, S8636), 2 mM L-glutamine (Sigma, G7513), 1000 U/ml leukemia inhibitory factor (LIF, Millipore, ESG1107), and penicillin/streptomycin (Gibco, 15140–122) at 37 °C with 5% CO2.
The commercial ESGRO-2i Medium (Merck-Millipore, SF-016-200) was also used when necessary36 (link). We found that in 2i medium, Adnp−/− ESCs adopted morphology indistinguishable to that of control ESCs, and maintain self-renewal capacity for more than 20 passages that we tested.
Sun X., Peng X., Cao Y., Zhou Y, & Sun Y. (2020). ADNP promotes neural differentiation by modulating Wnt/β-catenin signaling. Nature Communications, 11, 2984.
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3

Cdx2-Inducible Mouse ES Cell Line

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The Tet-inducible-Cdx2-Flag ES cell line (iCdx2 cells) was obtained from NIA mouse ES cell bank27 (link). iCdx2 cells were maintained on feeder cells in DMEM supplemented with 15% fetal bovine serum (Gibco, Thermo Fisher Scientific), 1% non-essential amino acids, 2 mM l-glutamine, 1,000 units of mLIF (EMD Millipore), 0.1 mM β-mercaptoethanol (Sigma), antibiotics and 2 μg/ml doxycycline (Sigma). When starting differentiation44 (link), iCdx2 cells were selectively seeded on gelatin-coated plates after stepwise elimination of the feeder MEF, then Cdx2 overexpression was induced by removing doxycycline. The cells were split at day 1 after doxycycline withdrawal and cell samples were collected in the following days.
Mouse ES cells in 2i/L were cultured in commercially available 2i medium kit (Milipore, SF016-200). a2i/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), 1X Pen/Strep (Invitrogen), 1000 units of LIF (Milipore), 1.5 μM CGP77675 (Tocris) and 3 μM CHIR99021 (Tocris). PKCi/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), PenStrep (Invitrogen), 1000 unites of mLIF (Milipore) and 5 μM Gö6976 (Tocris).
Li Z., Zhao S., Nelakanti R.V., Lin K., Wu T.P., Alderman MH I.I.I., Guo C., Wang P., Zhang M., Min W., Jiang Z., Wang Y., Li H, & Xiao A.Z. (2020). N(6)-methyladenine in DNA antagonizes SATB1 in early development. Nature, 583(7817), 625-630.
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4

Embryonic Stem Cell Differentiation

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E14 mouse embryonic stem cells were cultured in SL conditions as described above. Upon passage, cells were resuspended in the following media depending on the condition studied. Commercial 2i media containing LIF (Millipore, SF016–200) was used for BL, GL, 2iL, and ML experiments. For 2iL, all components were used according to the manufacturer’s recommendations. For GL and ML conditions, only the GSK3B inhibitor or MEK1/2 inhibitor was added, respectively. For the BL condition, no inhibitors were added. For the No condition, commercial 2i media without LIF (Millipore, SF002–100) was used with no inhibitors added. After 24 hours, the cells were washed with 1x DPBS and the media was changed. 48 hours after the initial media switch, the cells were collected using 0.25% trypsin-EDTA, quenched using serum containing media, washed in 1x DPBS and finally resuspended in 1x DPBS. The sample was then split in half. One half was resuspended in 200 µL of DPBS for genomic DNA extraction, as described above. The other half was resuspended in 500 µL of TRIzol reagent (Invitrogen, 15596018) and total RNA was extracted according to the manufacturer’s recommendations. Experiments for each condition were performed in triplicate.
Chialastri A., Sarkar S., Schauer E.E., Lamba S, & Dey S.S. (2023). Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity. bioRxiv.
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5

Generation of Inducible dCas9-expressing ESC Line

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To generate an inducible dCas9 expressing ESC cell line, we inserted tetracycline inducible dCas9-mCherry into the genome of a Nanog-Venus-mESC line using the PiggyBac transposon system (Ding et al., 2005 (link); Filipczyk et al., 2013 (link)) (Figure 1E). Nanog-Venus-mESC line is originally created by Filipczyk et al. from an R1 mouse ESC background which are male cells (R1/E (ATCC SCRC-1036). Cells were transfected with a tet-on dCas9 plasmid (pSLQ1942) and PiggyBac transposase using the Turbofect transfection reagent following the manufacturer’s instructions (R0531, ThermoFisher Scientific). A clonal line was generated by manually selecting an mCherry positive colony and expanding the colony in ESGRO-2i medium (SF016–200, Millipore). Addition of 1 mg/mL doxycyclin to the medium of the Nanog-Venus/dCas9-mCherry line resulted in a 65-fold increase in the dCas9-mCherry protein signal while the Nanog-Venus protein signal did not change (Figures S5A and S5B). A similar strategy was used to infect Nanog-Venus line with destabilized dCas9 vector, pSLQ2470, to generate the Nanog-Venus/ddCas9-mCherry. ddCas9-mCherry construct has a Destabilizing Domain at the N-terminal followed by dCas9 fused to mCherry through a P2A peptide.
Shariati S.A., Dominguez A., Xie S., Wernig M., Qi L.S, & Skotheim J.M. (2019). Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9. Molecular cell, 74(3), 622-633.e4.
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6

Cdx2-Inducible Mouse ES Cell Line

Cited 1 time
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The Tet-inducible-Cdx2-Flag ES cell line (iCdx2 cells) was obtained from NIA mouse ES cell bank27 (link). iCdx2 cells were maintained on feeder cells in DMEM supplemented with 15% fetal bovine serum (Gibco, Thermo Fisher Scientific), 1% non-essential amino acids, 2 mM l-glutamine, 1,000 units of mLIF (EMD Millipore), 0.1 mM β-mercaptoethanol (Sigma), antibiotics and 2 μg/ml doxycycline (Sigma). When starting differentiation44 (link), iCdx2 cells were selectively seeded on gelatin-coated plates after stepwise elimination of the feeder MEF, then Cdx2 overexpression was induced by removing doxycycline. The cells were split at day 1 after doxycycline withdrawal and cell samples were collected in the following days.
Mouse ES cells in 2i/L were cultured in commercially available 2i medium kit (Milipore, SF016-200). a2i/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), 1X Pen/Strep (Invitrogen), 1000 units of LIF (Milipore), 1.5 μM CGP77675 (Tocris) and 3 μM CHIR99021 (Tocris). PKCi/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), PenStrep (Invitrogen), 1000 unites of mLIF (Milipore) and 5 μM Gö6976 (Tocris).
Li Z., Zhao S., Nelakanti R.V., Lin K., Wu T.P., Alderman MH I.I.I., Guo C., Wang P., Zhang M., Min W., Jiang Z., Wang Y., Li H, & Xiao A.Z. (2020). N(6)-methyladenine in DNA antagonizes SATB1 in early development. Nature, 583(7817), 625-630.
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7

Lentiviral Transduction of Nanog-Venus/dCas9-mCherry ESCs

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Lentiviral particles containing sgRNA expression plasmids were generated by transfecting HEK293T cells with sgRNA plasmids, and with standard packaging constructs using the Turbofect transfection reagent (R0531, ThermoFisher Scientific) as previously described (Schwarz et al., 2018 (link)). One day after transfection, the HEK293T cell media was changed from DMEM/FBS to 2i (SF016–200, Millipore). The viral particles in the 2i media were collected after 48 h, centrifuged, and filtered (0.45-mm syringe filter). The particles were then added to media of the Nanog-Venus/dCas9-mCherry ESC line. The sgRNA expressing cells were selected using puromycin. The expression of sgRNAs was also visually confirmed by microscopy of BFP expression. Table S1 shows the sequences of all the sgRNAs used in this study.
Shariati S.A., Dominguez A., Xie S., Wernig M., Qi L.S, & Skotheim J.M. (2019). Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9. Molecular cell, 74(3), 622-633.e4.
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