title: RNA-seq of human fibroblast 1b
design: The NPCs were obtained through the Tau Consortium of the Neural Stem Cell Institute (www.neuralsci.org/ tau), cells were prepared with Geltrex (1:100 dilution) and seeded with Neurobasal medium ([-] L-glutamine) supplemented with B-27 (50X), AAS (100X) and Glutamax (100X), in addition, the growth factors bFGF (20 ng/ ml), EGF (20 ng/ ml) and LIF (10ng/ ml), to promote cell growth and division. RNA extraction, sequencing, processing and analysis. Once confluency was reached (80-100%), the RNA was extracted. Cells were mechanically detached from the cage and resuspended in RNAlater. Subsequently, they were stored at -80 C to be preserved. Afterwards, the cells were sent frozen to the FEMSA Biotechnology Center, TEC-Base Laboratory of the Tecnolgico de Monterrey for RNA isolation, library preparation and sequencing. RNA-sequencing & processing. Cells were centrifuged and resuspended in lysis buffer supplemented with 1% BME and RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). RNA concentration was measured with a Qubit RNA HS assay kit (Invitrogen, Carlsbad, CA, United States), while its purity was measured with a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE). To assess RNA integrity, all samples were analyzed with the Agilent RNA 6000 Pico Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The libraries were prepared with TruSeq Stranded Total RNA Library Prep in a Ribo-Zero Gold (Illumina, San Diego CA, United States), quantified with Qubit dsDNA HS assay kit (Invitrogen, Carlsbad, CA, United States), the size of the libraries was analyzed on a QSep 400 (BiOptic, New Taipei City, Taiwan) and sequencing was carried out on a NovaSeq 6000 using an S1 flow cell in a paired-end read configuration at 110 bp.