title: TBL
organism: Homo sapiens
description: Protocols: NONE Human H1 ESC was cultured in six-well culture plates (Nunc) coated with Matrigel (BD Biosciences, Canada) with PSCeasy medium (Beijing Cellapy Biotechnology, China). In initial experiments, cells were cultured on knockout serum replacement MEF-CM which contained recombinant FGF2 (4 ng/mL). For the differentiation experiment, the medium was changed again to one lacking FGF2 but containing BMP4 (10 ng/mL) and the ALK4/5/7 inhibitor A83-01 (1μM), and the FGF2-signaling inhibitor PD173074 (0.1μM). Control cultures continued to be grown in the presence of FGF2 and the absence of BMP4 and the inhibitors. The medium in all wells was changed daily. Cells were routinely passage at a 1:6 ratio every 5-6 d. Briefly, colonies were broken into small clumps of relatively uniform size with the Stempro EZpassage (Invitrogen) cutting tool according to the manufacturer's instructions. Total RNA was extracted with Trizol (Tiangen, Beijing) and assessed with Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer (Invitrogen). Total RNA samples that meet the following requirements were used in subsequent experiments: RNA integrity number (RIN) > 7.0 and a 28S:18S ratio > 1.8. RNA-seq libraries were generated and sequenced by CapitalBio Technology (Beijing, China). The triplicate samples of all assays were constructed an independent library, and do the following sequencing and analysis. The NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) was used to construct the libraries for sequencing. NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB) kit was used to enrich the poly(A) tailed mRNA molecules from 1 μg total RNA. The mRNA was fragmented into ~200 base pair pieces. The first-strand cDNA was synthesized from the mRNA fragments reverse transcriptase and random hexamer primers, and then the second-strand cDNA was synthesized using DNA polymerase I and RNaseH. The end of the cDNA fragment was subjected to an end repair process that included the addition of a single “A” base, followed by ligation of the adapters. Products were purified and enriched by polymerase chain reaction (PCR) to amplify the library DNA. The final libraries were quantified using KAPA Library Quantification kit (KAPA Biosystems, South Africa) and an Agilent 2100 Bioanalyzer.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-11-16T09:23:10Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-11-06T10:54:32Z
attributes.tag: External Id
attributes.value: SAMEA7534181
attributes.tag: INSDC center name
attributes.value: 1. Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou, China; 2. Academy of Medical Sciences of Zhengzhou University Translational Medicine platform, Zhengzhou University, No.100 Science Avenue, Zhengzhou, China;
attributes.tag: INSDC first public
attributes.value: 2020-11-16T09:23:10Z
attributes.tag: INSDC last update
attributes.value: 2020-11-06T10:54:32Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9738:TBL
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: trophoblast-like cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo stage
attributes.tag: disease
attributes.value: normal
attributes.tag: sample name
attributes.value: E-MTAB-9738:TBL
attributes.tag: scientific_name
attributes.value: Homo sapiens