Polypeptide gene cDNA, vector

cDNAs were synthesized from mRNA isolated from mantle epithelial tissue of pearl oyster (Pinctada fucata) using reverse transcriptase and joined to a vector to yield a cDNA library. A cDNA inserted into this cDNA library was made to be expressed in Escherichia coli, for example, to give a polypeptide corresponding to said cDNA. This polypeptide serves as a novel ingredient of cosmetic compositions, for example.

Conventional hiPSCs (CRISPRi (GEN1C clone)) were maintained on Matrigel-coated dishes (Corning) in mTeSR1 medium (Stem Cell Technologies) and passaged using Gentle Cell Dissociation Reagent (Stem Cell Technologies). For maintenance, cells were subcultured every 4-5 days. Overexpression of DDX6 in hiPSC GEN1C cells (GEN1C DDX6 OE) was achieved by infection with a lentiviral vector expressing the wt DDX6 cDNA (Vector Builder).

For the TGFβi assay, 40,000 GEN1C hiPSCs were seeded in each well of a 24-well plate coated with Matrigel in mTeSR1 medium (Stem Cell Technologies) supplemented with 10 μM Y-27632 (Axon Medchem). 24 hours after seeding, the mTeSR1 medium was replaced with mTeSR1 + 1 μM A8301 (Stemgent). Cells were incubated in differentiation media for 48 hours for the TGFβi condition. Media were then replaced with mTeSR1 and incubated for an additional 24 hours prior to FACS analysis for OCT4-GFP expression.

RNA sequencing libraries were prepared using the NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina (Cat#: E7420). RNA input for library construction was 50 ng in total. Libraries were amplified for 14 cycles. After library construction, samples were validated using the Tapestation 2200 system and the high-sensitivity ScreenTape D1000 kit. Libraries were quantified using the Kapa Biosystems Library Quantification Kit (catalog number KK4828) and the BioRad CFX96 instrument.