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Sample GSM3665815

Query DataSets for GSM3665815

Status

Public on Oct 01, 2019

Title

XEN-VI rep1

Sample type

SRA

Source name

Embryonic cells

Organism

Sus scrofa

Characteristics

sample type: eXtraEmbryonic eNdoderm stem (XEN) cells from primitive endoderm of an in vivo blastocyst

Treatment protocol

The pXEN cells were differentiated by means of embryoid body (EB) formation and treatment with small molecules and factors as previously described [#ref]. pXEN cells were dissociated as clumps, washed, and resuspended in medium (DMEM/F12 plus 15% FBS) as hanging drops on the lid of a 60mm dish, and cultured for 5 days, during which time spheroids were formed. To direct pXEN cells differentiation into either visceral endoderm (VE) or parietal endoderm (PE), accutase-dissociated single cells (2 x 105 cells per cm2) were seeded onto a laminin- or fibronectin-coated 6 well plate in N2B27 medium supplemented with the respective differentiation factors and/or chemicals. For example, for differentiation into VE, the cells were treated with CHIR99021(10 μM, stem cell biotech) and BMP4 (50 ng/mL, R&D) for activating Wnt/β-catenin pathway; for differentiation into PE, Folskolin (50 μM) and dbcAMP (1 mM) for activating the cyclic adenosine monophosphate (cAMP) signaling pathway were utilized; as per previous publications [#ref]. Differentiation medium was replaced every two days, and cells were processed for analysis on day 12.

Growth protocol

Embryonic explants and XEN cells were cultured on a feeder layer of early passage (n=3) CF-1 mouse embryonic fibroblasts (MEF) cells mitotically inactivated by treatment with mitomycin-C (3 hr, 10 μg/mL). A day before seeding the embryos or XEN cells, the feeders were plated in MEF medium (high-glucose Dulbecco's modified Eagle medium, DMEM (Gibco, Grand Island, NY) supplemented with 10% (v/v) FBS (HyClone, Logan, UT)) on 0.1% (v/v) gelatin-coated four-well plates (Nunclon) at a density of 3-5 × 105 cells per cm2. At least 2 hr before the start of the experiment, the MEF medium was aspirated and replaced with ‘standard ES medium’ which included DMEM/ Nutrient Mixture Ham's F12 (DMEM/F-12, Gibco) supplemented with 15% ES-qualified fetal calf serum (FCS, HyClone), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 units/mL penicillin-streptomycin, 0.1 mM 2-β-mercaptoethanol, 1% non-essential amino acids (NEAA) (all from Gibco), with various combination of growth factors; 10 ng/mL human recombinant leukemia inhibitory factor (hrLIF; Milipore, Bedford, MA) and 10 ng/mL human recombinant basic fibroblast growth factor (hrbFGF; R&D Systems, Minneapolis, MN). Other media combinations that were tested include RPMI 1640 or N2B27 serum free medium (1:1 ratio of DMEM/F12 and Neurobasal medium plus N2 and B27) (all from Gibco), with a com bination of LIF and/or bFGF, or 25 ng/mL human recombinant fibroblast growth factor 4 (hrFGF4; R&D Systems) and 1 μg/mL heparin. Following initial plating, attachment and outgrowth development, the medium was refreshed on d 3, followed by media exchange every 2 days. After 7-8 days of culture, the primary outgrowths were mechanically dissociated into small clumps, and transferred onto fresh feeders for passaging. The pXEN cells were cultured at 38.5°C in 5% O2 and 5% CO2, with the culture medium being refreshed every other day and passaged at 1:20 every 7-8 days. Cells were passaged as clumps by gentle pipetting following 10 min digestion with Accutase (Gibco).

Extracted molecule

polyA RNA

Extraction protocol

For isolation of genomic DNA (gDNA) from cells and tissues, the QIAamp mini DNA Kit (Qiagen) was used according to the manufacturers’ instructions. Total RNA was isolated using Trizol plus RNeasy mini kit (Qiagen) and mRNA from individual blastocysts was extracted using the Dynabeads mRNA Direct Kit (Dynal Asa, Oslo, Norway).
Synthesis of cDNA was performed using a High Capacity cDNA Reverse transcription kit (Applied Biosystems; ABI, Foster City, CA) according to the manufacturers’ instructions. The QIAseq FX Single Cell RNA Library kit (Qiagen) was used for Illumina library preparation and transcriptomics analysis.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Description

eXtraEmbryonic eNdoderm stem (XEN) cells from primitive endoderm of an in vivo blastocyst

Data processing

RNA-seq reads were mapped to the pig reference genome (Sscrofa11.1) using HISAT21 (version 2.0.4) with parameters “hisat2 --sensitive --no-discordant --no-mixed -I 1 -X 1000” and to the reference cDNA sequence using Bowtie22 with parameters “bowtie2 -q --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200”.
Then the expression levels of each gene were calculated by the fragments per kilobase of exons per million fragments mapped (FPKM) using RSEM3 with parameters “rsem-calculate-expression --paired-end -p 8” based on the result of Bowtie2.
The final expression matrix was subjected to hierarchical clustering using R software.
Development stage (PE, PrE, TE, VE and EPI)-specific genes were selected to do the subsequent analyses. They were mapped to the final expression matrix to do the PCA and heatmap analysis with R software.
FPKMs of 13,825 protein coding genes detected by RNA-seq
Genome_build: Sscrofa11.1
Supplementary_files_format_and_content: tab-delimited Excel files include FPKM values for each sample

Submission date

Mar 11, 2019

Last update date

Oct 01, 2019

Contact name

CNSA CNGB

Organization name

BGI

Street address

BGI

City

shenzhen