|
| Status |
Public on Aug 13, 2020 |
| Title |
RNA-seq_E35A-4d-Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
3T3-L1
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse fibroblasts
histone: E35A histone
time point: differentiation day 4
cell line: 3T3-L1
|
| Treatment protocol |
3T3-L1 cells expressing inducible FLAG-tagged wild-type or mutant (E35A, S36A) histone H2B were seeded in 6-well plates. Differentiation from preadipocytes into adipocytes was induced in contact-inhibited cells by the addition of MDI cocktail [0.25 mM IBMX (3-isobutyl-1-methylxanthine; Calbiochem, 410957), 1 mM dexamethasone (Sigma, D4902), and 10 mg/mL insulin (Sigma, I-5500)]. The cells were treated with MDI cocktail for 2 days and then cultured in medium containing 10 mg/mL insulin for the indicated time points before collection.
|
| Growth protocol |
3T3-L1 cells were obtained from the American Type Cell Culture (ATCC, CL-173) and were regularly verified as mycoplasma-free. They were cultured in DMEM (Cellgro, 10-017-CM) supplemented with 10% fetal bovine serum (Sigma, F8067) and 1% penicillin/streptomycin.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
3T3-L1 cells were collected at the indicated time points and total RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer’s instructions.
The total RNA was then enriched for polyA+ RNA using Dynabeads Oligo(dT)25 (Invitrogen). The polyA+ RNA was then used to generate strand-specific RNA-seq libraries as described previously. The RNA-seq libraries were subjected to QC analyses (i.e., number of PCR cycles required to amplify each library, the final library yield, and the size distribution of final library DNA fragments) and sequenced using an Illumina HiSeq 2000
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNA-seq in 3T3-L1 cells ectopically expressing E35A histone H2B at differentiation day 4
|
| Data processing |
The raw data were subjected to QC analyses using the FastQC tool. The reads were then mapped to mouse genome (mm10) using the spliced reader aligner TopHat version 2.0.13. Transcriptome assembly was performed using cufflinks v.2.2.1 with default parameters48. The transcripts were merged into two distinct, non-overlapping sets using cuffmerge, followed by cuffdiff to call the differentially regulated transcripts. The significantly (p <0.05) regulated genes from cells expressing the H2B mutants S36A or E35A were compared to wild-type at the indicated time points.
Supplementary_files_format_and_content: Gene-level differential expression file
|
| |
|
| Submission date |
Aug 20, 2019 |
| Last update date |
Aug 13, 2020 |
| Contact name |
W. Lee Kraus |
| E-mail(s) |
lee.kraus@utsouthwestern.edu
|
| Organization name |
UT Southwestern Medical Center
|
| Street address |
5323 Harry Hines Blvd.
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-8511 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE136055 |
Functional Interplay between Histone H2B ADP-Ribosylation and Phosphorylation Controls Adipogenesis |
|
| Relations |
| BioSample |
SAMN12608555 |
| SRA |
SRX6743619 |
