|
| Status |
Public on Jun 26, 2018 |
| Title |
mSVF BAT siKO d3 3 |
| Sample type |
SRA |
| |
|
| Source name |
brown adipose tissue
|
| Organism |
Mus musculus |
| Characteristics |
tissue: brown adipose tissue
strain: C57BL/6
cell type: stromal vascular fraction
treatment: day3
sirna: non-targeting control siRNA
|
| Treatment protocol |
Short interfering RNAs (siRNAs; siH19, non-targeting Control, siCtrl) were delivered into BAT or WAT SVFs by Amaxa nucleofection (Lonza Bioscience) according to manufacturer‘s recommendations. Cells were utilized 48–72 h after transfection. To induce commitment of SVFs into mature adipocytes, freshly prepared 0.05 % Insulin, 0.005 % Dexmethasone, 0.001 % Rosiglitazone and 0.05 % 3-Isobutyl-1-methylxanthin (IBMX; WAT and BAT) or 0.1 % Indomethazine, 0.001 % Triiodothyronine (BAT) in growth medium (induction medium) were added to confluent cells. After 48 h of induction, differentiation was replaced with freshly prepared 0.001 % Rosiglitazone (WAT) or 0.001 % Triiodothyronine (BAT) in growth medium (differentiation medium). RNA was iisolated at day3 of differentiation.
|
| Growth protocol |
Isolated BAT or WAT SVFs were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.5 g/l glucose, 20 % fetal calf serum (FCS), 20 mM HEPES, sodium pyruvate, L-glutamine, non-essential amino acids and 1 % gentamycin and cultured at 37° C with 5 % CO2. Media was changed every day until cells had reached 80 % confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the RNeasy mini kit (Qiagen) according to manufacturer’s protocols for total RNA isolation
Libraries were prepared according to NEB´s instructions accompanying the NEBNExt Ultra II Directional RNA Library Prep Kit (Part# E7760S). 20ng of total RNA was enriched for the Poly-A mRNA and reverse transcribed to double-stranded cDNA followed by 14 cycles of PCR. Sequencing was performed using 1,3pM hybridization to a 2x100 paired end flow cell.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
MB-3_6
|
| Data processing |
Quality control of the raw FASTQ files using FastQC_v0.10.1
Alignment of reads against reference genome using tophat-2.0.10.Linux_x86_64
Alignment of RNA-Seq reads with the STAR aligner
Call of FPKM values per transcript using cufflinks-2.2.1.Linux_x86_64
Differential expression analysis on gene level using DESeq2 (R-3.2.2)
Genome_build: GRCm38.p5; Ensmbl Build 87
|
| |
|
| Submission date |
Jun 25, 2018 |
| Last update date |
Jun 26, 2018 |
| Contact name |
Jan-Wilhelm Kornfeld |
| E-mail(s) |
[email protected]
|
| Phone |
+45-9350 7481
|
| Organization name |
Max-Planck-Institute for Metabolism Research
|
| Lab |
Noncoding RNA and Energy Homeostasis
|
| Street address |
Gleueler Strasse 50
|
| City |
Cologne |
| State/province |
NRW |
| ZIP/Postal code |
50931 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE116226 |
Regulation of brown fat homeostasis by H19 lincRNA [II] |
| GSE116227 |
Regulation of brown fat homeostasis by H19 lincRNA |
|
| Relations |
| BioSample |
SAMN09479648 |
| SRA |
SRX4292301 |
