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Sample GSM5244397

Query DataSets for GSM5244397

Status

Public on Mar 29, 2022

Title

H9_HEP_Cont_0DAH_rep3

Sample type

SRA

Source name

Hepatocyte-like cells derived from H9 human embryonic stem cells

Organism

Homo sapiens

Characteristics

condition: 37degreeC
derived tissue: Human hepatocyte-like cells
time point: 0DAH

Treatment protocol

On day 14, cells were incubated at 37or 39˚C, and half the total amount of the medium was changed every day.

Growth protocol

Before inducing differentiation, we coated a cell-culture dish with 0.1% gelatin in phosphate-buffered saline (PBS, Thermo Fisher Scientific) for 30 min at 25˚C. We then aspirated the gelatin solution and introduced a DMEM/F12 medium (Sigma-Aldrich) onto the culture dish for serum coating at 37˚C for 24 h. The medium was supplemented with 10% (v/v) fetal bovine serum (Cell Culture Bioscience, Tokyo, Japan), penicillin/streptomycin (Wako), and 100 µM β-mercaptoethanol (Sigma-Aldrich). The coated dish was then rinsed with fresh medium. Cultured hPSCs were washed with PBS and treated with TryPLE Express at 37˚C for 5 min, followed by the addition of basal medium and the transfer of the cell suspension into a 15 mL tube to induce endoderm differentiation. Cells were centrifuged at 200 × g for 3 min, the supernatant was removed, and then the cells were resuspended in mTeSR-1 medium supplemented with 10 µM Y27632 and 100 ng mL−1 activin A (R&D Systems, Minneapolis, MN, USA), plated on a serum-coated culture dish, and cultured in a humidified incubator at 37˚C with 5% CO2 for 24 h. At the end of day 1, the medium was replaced with fresh mTeSR-1 medium supplemented with 10 µM Y27632 and 100 ng mL−1 activin A and cultured for another 24 h. On day 2, the medium was replaced with mTeSR-1 medium supplemented with 10 µM Y27632, 100 ng mL−1 activin A, 10 ng mL−1 BMP-4 (R&D Systems), 10 µM LY294002 (Cayman Chemical, Ann Arbor, MI, USA), and 3 µM CHIR99021 (Stemgent, Cambridge, MA, USA), and cells were incubated for 24 h. On day 3, the medium was replaced with mTeSR-1 medium supplemented with 10 µM Y27632, 100 ng mL−1 activin A, 10 ng mL−1 BMP-4, and 10 µM LY294002, and cells were incubated for 24 h. On day 4, the medium was replaced with Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific) supplemented with B-27 (Thermo Fisher Scientific), 100 ng mL−1 activin A, and 100 ng mL−1 basic FGF, and cells were incubated for 24 h. To induce ADE specification, cells were treated with RPMI medium supplemented with 50 ng mL−1 activin A, with daily medium changes for three days. Cells were then treated with RPMI medium supplemented with 20 ng mL−1 BMP-4 and 10 ng mL−1 FGF-10 (R&D systems), with daily medium changes for four days. On day 12, the medium was replaced with hepatocyte-maturation medium (hepatocyte basal medium (Lonza, Basel, Switzerland) supplemented with 30 ng mL−1 oncostatin M (R&D Systems), 50 ng mL−1 HGF (PeproTech, Rocky Hill, NJ), and 25 mM HEPES (Wako) to induce maturation of the differentiated hepatocytes. On day 14, cells were incubated at 37, 39, or 42˚C, and half the total amount of the medium was changed every day.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from hepatocyte-like cells derived from H9 human embryonic stem cells using the RNeasy mini kit (QIAGEN).
To prepare the cDNA library, 10 ng of total RNA was diluted with 9 µL of RNase free water, then mixed with VN primer (Oxford NANOPORE Technologies, UK), 1 µL of 10 mM dNTPs (New England Biolabs Inc. Ipswich, Massachusetts, USA), and incubated at 65˚C for 5 min as the RNA solution. Separately, 4 µL of 5x RT Buffer (Thermo Fisher Scientific), 1 µL of RNaseOUT (Thermo Fisher Scientific), 1 µL of Nuclease-free water and 2 µL of Strand-Switching Primer (Oxford NANOPORE Technologies) was mixed as the strand-switching buffer. The two solutions were mixed at 42˚C for 2 min; then, 1 µL of Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) was added, incubated at 42˚C for 90 min, 85˚C for 5 min, and stored at 4˚C until use as the cDNA library. Oxford NANOPORE Technologies Nanopore MinION Flowcell v.9.4.1.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Description

mRNA-seq data from 37˚C-treated cells at 0DAH, replicate 3

Data processing

Base-calling and demultiplexing were performed with EPI2ME software (Oxford NANOPORE Technologies).
Failed reads were discarded and Fast5 files were converted into FASTQ and FASTA files using EPI2ME software.
Generated FASTQ files were loaded to BioJupies for alignment with human genomes and annotation.
the counts were analysed using TCC in R Bioconducter. Briefly, the counted data set was introduced into the TCC-GUI package.
Genome_build: human_matrix.h5 v2
Supplementary_files_format_and_content: Gene counts were normalized using TMM (Trimmed mean of M values) methods in the edgeR package with the parameters: Number of Iterations = 3; FDR < 0.1; Elimination of Potential DEGs = 0.05.

Submission date

Apr 16, 2021

Last update date

Mar 29, 2022

Contact name

Ken-ichiro Kamei

E-mail(s)

[email protected]

Phone

+81-757539774

Organization name

Kyoto University