 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 17, 2021 |
| Title |
RNA_Med1_8wk_AKO_BAT |
| Sample type |
SRA |
| |
|
| Source name |
RNA_Med1_8wk_AKO_BAT
|
| Organism |
Mus musculus |
| Characteristics |
strain background: 129S4/SvJae - C57BL/6 mixed
genotype/variation: Med1f/f;Adipoq-Cre
tissue: Brown adipose tissue
|
| Treatment protocol |
ChIP-seq: For adipogenesis assay, cells were plated in growth medium (DMEM plus 10% FBS) at a density of 1.8x106 cells per 15-cm dish 4 days before induction of adipogenesis. At D0, confluent cells were induced with 0.02 μM insulin, 1 nM T3, 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX), 2 μg/ml dexamethasone, and 0.125mM indomethacin. At D2, D4, and D6, the cells were replenished with medium containing 10% FBS, 0.02 μM insulin, and 1 nM T3.
|
| Growth protocol |
ChIP-seq: Primary brown preadipocytes were isolated from interscapular BAT of newborn Med1f/f;CreER mice) and immortalized with SV40T-expressing retroviruses generated from the pBabeneo-large T plasmid
|
| Extracted molecule |
total RNA |
| Extraction protocol |
ChIP-seq: Cells were cross-linked with 1.5% formaldehyde for 10 min and quenched by 125 mM glycine for 10 min. 10 million fixed cells were resuspended in ice cold buffer containing 5mM PIPES, pH 7.5, 85mM KCl, 1% NP-40 and protease inhibitors, incubated on ice for 15 min, and centrifuged at 500 g for 5 min at 4°C. Nuclei were resuspended with 1mL buffer containing 50mM Tris-HCl, pH8.0, 10mM EDTA, 0.1% SDS and protease inhibitors, and subjected to sonication. Sheared chromatin was clarified by centrifugation at 13,000 g for 10 min at 4°C. The supernatant was transferred to a new tube and further supplemented with 1% Triton-X100, 0.1% sodium deoxycholate and protease inhibitors. 2% of the mixture was set aside as input and 20ng of spike-in chromatin (Active Motif, #53083) was added to the rest. For each ChIP, 4 - 10 µg of target antibodies and 2μg of spike-in antibody (anti-H2Av, Active Motif, #61686) were added and incubated on a rotator at 4°C overnight. ChIP samples were added with 50μL prewashed protein A Dynabeads (ThermoFisher) and incubated for 3h at 4°C. Beads were then collected on a magnetic rack and washed twice with 1mL cold RIPA buffer, twice with 1mL cold RIPA buffer containing 300mM NaCl, twice with 1mL cold LiCl buffer and once with PBS. Beads were then eluted with 100 μL buffer containing 0.1M NaHCO3, 1% SDS, and 20μg Proteinase K at 65°C overnight. Input sample volumes were adjusted to 100μL with the elution buffer. Samples were then purified using QIAquick PCR purification kit (Qiagen) and eluted in 30 μL 10mM Tris-HCl.
ChIP-seq: Libraries were prepared according to NEB (Illumina kit) instructions. DNA were used to construct libraries using NEBNext® Ultra™ II DNA Library Prep kit with AMPure XP magnetic beads (Beckman Coulter).
ATAC-Seq: 5 × 10^4 freshly collected cells were aliquoted into a new tube and spun down at 500 × g for 5 min at 4 °C. The cell pellet was incubated in 50 µl of ATAC-RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin (Promega) on ice for 3 min and was washed out with 1 ml of ATAC-RSB buffer containing 0.1% Tween-20. Nuclei were collected by centrifugation at 500 × g for 10 min at 4 °C, then resuspended in 50 µl of transposition reaction buffer (25 µl 2x Tagment DNA buffer, 2.5 µl transposase (100 nM final; Illumina), 16.5 µl PBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-20, 5 µl H2O). The reaction was incubated at 37 °C for 30 min with mixing (1000 r.p.m.) and directly subjected to DNA purification using the MinElute Reaction Cleanup Kit (Qiagen) according to the manufacturer’s instructions.
ATAC-Seq: DNA was amplified with PCR using Nextera i7- and i5-index primers for Illumina protocols
RNA-Seq: Total RNA was subjected to mRNA purification using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490).
RNA-Seq: Isolated mRNAs were reverse-transcribed into double stranded cDNA and subjected to sequencing library construction using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina (NEB, E7770) according to the manufacturer’s instructions.
nascent RNA-Seq: 10^6 cells were labeled with 0.5 mM ethylene uridine for 30 min at 37 °C. After RNA extraction, 1 μg total RNA was depleted of rRNA using the NEBNext rRNA Depletion kit (NEB). To capture nascent RNA, the sample was biotinylated by the Click-iT Nascent RNA Capture Kit (ThermoFisher, C10365) according to the manufacturer’s instructions.
nascent RNA-Seq: Double-stranded cDNAs were synthesized using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). Library construction was done using NEBNext Ultra™ II DNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Med1_RNA_8wk_AKO_BAT_S42_L007
|
| Data processing |
Basecalls performed using Bcl2fastq.
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie2.
Peaks were called using SICER with following configuration: For ChIP-seq of histone modifications: Windows (200), Gaps (200), FDR (1e-3); For ChIP-Seq of factors: Windows (50), Gaps (50), FDR (1e-3)
Single or paired-end reads of RNA-seq are aligned to the mm9 genome assembly using STAR.
SICER v1.1 was used to calculate the reads per kilo base per million (RPKM) on exonic regions as expression measurements for each gene.
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using in-house script and the scores represent RPKM; tab-delimited text files include FPKM values for each gene.
|
| |
|
| Submission date |
Nov 01, 2020 |
| Last update date |
Mar 20, 2021 |
| Contact name |
Kai Ge |
| E-mail(s) |
kai.ge@nih.gov
|
| Phone |
301-451-1998
|
| Organization name |
NIH
|
| Department |
NIDDK
|
| Street address |
50 South Dr Rm 4154
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE160605 |
MED1 is a lipogenesis co-activator required for postnatal adipose expansion |
|
| Relations |
| BioSample |
SAMN16621789 |
| SRA |
SRX9415844 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4875860_Med1_RNA_8wk_AKO_BAT_sorted_readcount_on_exon.txt.gz |
291.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
