|
| Status |
Public on Dec 01, 2018 |
| Title |
RNA-seq, BM-hMSC-TERT4 adipocyte diff 3d rep3 |
| Sample type |
SRA |
| |
|
| Source name |
BM-hMSC-TERT4 osteAdlast diff 3d
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BM-hMSC-TERT4
differentiation status: adipocyte
timepoint: 3d
chip antibody: n/a
|
| Treatment protocol |
Two days post confluency cells were induced to undergo osteoblast or adipocyte differentiation by switched to α-MEM supplemented with 10% FCS, 10 mM dexamethasone, 10 nM vitamin D, 10 mM β-glycerolphosphate, and 50 µg/ml ascorbic acid or to DMEM supplemented with 10% fetal serum, 10ug/mL insulin, 1µM rosiglitazone, 100 mM dexamethasone, and 500 µM isobutylmethylxanthine. Media was replaced on day 2, 4, 7, 9 and 11.
|
| Growth protocol |
Telomerase-immortalized human mesenchymal stromal cells of bone marrow (BM-hMSC-TERT4) or adipose tissue origin (AT-hMSC-TERT) were grown under standard cell culture conditions in α-MEM supplemented with 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin (P/S).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from 6-well dishes using TRI Reagent (Sigma) follwed by chloroform extraction and purification of RNA using Econo Spin columns (Epoch Life Sciences).
RNA-seq was performed according to manufacturer’s instructions (TruSeq 2, Illumina) using 2 µg RNA for preparation of cDNA libraries.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
RNA_3dAd_BM_rep3
GeneExpr_BM.txt
|
| Data processing |
Quality of all libraries were assessed using FastQC (http://www.bioinformatics.babraham.ac.uk /projects/fastqc/).
ChIP-seq and DNase-seq reads were aligned to the hg19 genome using STAR aligner (Dobin A, et al., Bioinformatics 29:15-21) with the following parameters: --outSJfilterIntronMaxVsReadN 0 --alignIntronMax 1 --alignSJDBoverhangMin 200, with all other parameters set at default. Only one read per position and read length was kept. RNA-seq reads were aligned to hg19 genome using STAR aligner and default settings. Only one read per position and read length was kept.
Regions enriched for open chromatin or MED1 recruitment were identified using the ENCODE IDR (LI Q H, et al. 2011. Annals of Applied Statistics, 5:1752-1779) pipeline and MACS2 peak caller (Zhang Y, et al. 2008. Genome Biology 9:R137). For ChIP-seq samples a pooled Inout control sample was used as control. Reproducible peaks between replicates for the single time points were merged using Homer with distance option -d given.
Gene expression was quantified using HOMER (Heinz S, et al. 2010. Molecular Cell 38: 576-589) with the options -count exons -condenseGenes -noCondensing -noadj. Normalization and differential expression analysis was performed using DEseq2 (Love M I, et al. 2014. Genome Biology 15: 550) taking Replicates and Time Points into the design matrix, excluding genes which were not detected in both cell lines. Log 2 fold changes and FDR values were calculated with respect to undifferentiated MSC.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph and bigWig files were created using HOMER for visualization of enhancer features in genome browser. Bed files contain reproducible genomic regions from IDR analysis of DNase1-seq, MED1 ChIP-seq and their overlap with and without promoters. Txt file contains non-adjusted gene expression values for eauch replicate and averaged normalized counts and standard errors for each time point as well as FDR and Log2FC with respect to undifferentiated MSC for both BM-hMSC-TERT4 and AT-hMSC-TERT4 cells.
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| |
|
| Submission date |
Apr 17, 2018 |
| Last update date |
Dec 01, 2018 |
| Contact name |
Susanne Mandrup |
| E-mail(s) |
s.mandrup@bmb.sdu.dk
|
| Phone |
+45 6550 2340
|
| Organization name |
University of Southern Denmark
|
| Department |
Biochemistry and Molecular Biology
|
| Street address |
Campusvej 55
|
| City |
Odense M |
| ZIP/Postal code |
5230 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL18460 |
| Series (1) |
| GSE113253 |
Osteogenesis depends on commissioning of a network of stem cell transcription factors that act as repressors of adipogenesis |
|
| Relations |
| BioSample |
SAMN08941782 |
| SRA |
SRX3946971 |
