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5 protocols using pou5f1

1

Profiling Pluripotency Genes in Germ Cell-Like Cells

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PGCLCs sorted or harvested from membranes were placed in 350 μL of RLT buffer (QIAGEN) and RNA was extracted using RNeasy micro kit (QIAGEN, 74004). cDNA was synthesized using SuperScript® II Reverse Transcriptase (Invitrogen, 18064-014). Real time quantitative PCR was performed using TaqMan® Universal PCR Master Mix (Applied Biosystems, 4304437) and the expression level of genes-of-interest were normalized to the expression of housekeeping gene GAPDH. The TaqMan probes used in this study include: GAPDH (Applied Biosystems, Hs99999905_m1), NANOS3 (Applied Biosystems, Hs00928455_s1), PRDM1 (Applied Biosystems, hs01068508_m1), TFAP2C (Applied Biosystems, Hs00231476_m1), POU5F1 (Applied Biosystems, Hs03005111_g1), SOX2 (Applied Biosystems, Hs01053049_s1), PRDM14 (Applied Biosystems, Hs01119056_m1).
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2

Profiling Pluripotency Genes in Germ Cell-Like Cells

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PGCLCs sorted or harvested from membranes were placed in 350 μL of RLT buffer (QIAGEN) and RNA was extracted using RNeasy micro kit (QIAGEN, 74004). cDNA was synthesized using SuperScript® II Reverse Transcriptase (Invitrogen, 18064-014). Real time quantitative PCR was performed using TaqMan® Universal PCR Master Mix (Applied Biosystems, 4304437) and the expression level of genes-of-interest were normalized to the expression of housekeeping gene GAPDH. The TaqMan probes used in this study include: GAPDH (Applied Biosystems, Hs99999905_m1), NANOS3 (Applied Biosystems, Hs00928455_s1), PRDM1 (Applied Biosystems, hs01068508_m1), TFAP2C (Applied Biosystems, Hs00231476_m1), POU5F1 (Applied Biosystems, Hs03005111_g1), SOX2 (Applied Biosystems, Hs01053049_s1), PRDM14 (Applied Biosystems, Hs01119056_m1).
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3

Stem Cell Characterization by qPCR

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RNA was isolated via QIAcube (serial number: 50946) and RNeasy Mini Kit protocol “Purification of total RNA from 5 × 106 − 1 × 107 animal cells (with QIAshreddder homogenization)” (Firmware Version FIW-50–001-J_FW_MB.hex and program version FIW-50–002-G_PLC_MB.prs or higher) following manufacturer’s instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosysystems, Cat. # 4368814) according to manufacturer’s instructions. Quantitative PCR with the Taqman primers POU5F1 (Thermofisher, Cat. # 4448892), PAX6 (Thermofisher, Cat. # 4448892), NES (Thermofisher, Cat. # 4448892), TUBB3 (Thermofisher, Cat. # 4448892), and OTX2 (Thermofisher, Cat. # 4448892) was carried out by a liquid handling robotic system and run via Quantstudio 12K Flex (serial number: 285880333).
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4

Stem Cell Characterization by qPCR

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RNA was isolated via QIAcube (serial number: 50946) and RNeasy Mini Kit protocol “Purification of total RNA from 5 × 106 − 1 × 107 animal cells (with QIAshreddder homogenization)” (Firmware Version FIW-50–001-J_FW_MB.hex and program version FIW-50–002-G_PLC_MB.prs or higher) following manufacturer’s instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosysystems, Cat. # 4368814) according to manufacturer’s instructions. Quantitative PCR with the Taqman primers POU5F1 (Thermofisher, Cat. # 4448892), PAX6 (Thermofisher, Cat. # 4448892), NES (Thermofisher, Cat. # 4448892), TUBB3 (Thermofisher, Cat. # 4448892), and OTX2 (Thermofisher, Cat. # 4448892) was carried out by a liquid handling robotic system and run via Quantstudio 12K Flex (serial number: 285880333).
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5

Transcriptional Profiling of Stem Cell Markers

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Cells were plated in 25 cm2-angled flasks at 1 × 10^6 cells of confluence, 48 h after treatments, cells were scraped from flasks to extract RNA; the extraction was performed with RNAeasy kit (QUIAGEN, Hilden Germany) according to manufacturer’s instructions. Next cDNA was obtained by retro-transcription assay using Revert Aid Synthesis Kit (Thermofisher, United States). For RT-PCR assays, taqman probes used for: (Hs00610060_m1) SFRP1; (Hs00277039_m1) CCND1; (Hs03986777_s1) FZD4; (Hs02387400_g1) NANOG; (Hs99999903_m1) ACTB; (Hs00153408_m1) MYC; (Hs04260367_gH) POU5F1; (Hs00610344_m1) AXIN2; (Hs00602736_s1) SOX2; (Hs99999901_s1) 18S, and (Hs01547250_m1) LEF1 genes were purchased from Thermofisher (Massachusetts, United States).
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