|
| Status |
Public on May 19, 2016 |
| Title |
double thymidine block at 4.5h |
| Sample type |
SRA |
| |
|
| Source name |
S-G2
|
| Organism |
Homo sapiens |
| Characteristics |
cell cycle: S-G2
cell line: HeLa cells
sample type: double thymidine block at 4.5h
|
| Treatment protocol |
Cells were subsequently treated with 2 mM thymidine (Sigma) for a total of 18 hrs, washed 2 times with 1xPBS, and supplemented with fresh complete media for 10 hrs. 2 mM thymidine was subsequently added for a second block of 18 hrs and washed as described previously. Mitotic block was performed by double thymidine arrest (as above) and release in fresh media for 3.5 hrs followed by addition of nocodazole 100 μM (Sigma) for 10 hrs. G1 block was performed by serum starvation for 72 hrs in DMEM containing 0.05% FBS.
|
| Growth protocol |
Cells were cultured in humidified incubators with 5% CO2. Cell cycle synchronization was adapted from the protocol of Whitfield et al. (Whitfield et al., 2002); ~ 750,000 log phase HeLa cells were plated in 15 cm dishes in complete media and allowed to attach for 16 hrs, reaching < 30% confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-Seq, cells (both adherent and detached) were harvested every 1.5 hrs for 30 hrs and frozen immediately for purification of total RNA.
RNA from synchronized cells was extracted with TRIZOL (Invitrogen), treated with DNAse I (Qiagen), and purified on RNAeasy columns. (Qiagen) according to the manufacturer’s protocol. RNA-seq libraries were robotically prepared with Illumina TruSeq Total RNA Sample Prep kits according to the manufacturer’s protocol
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
RNA-Seq reads were mapped to the human genome (build hg19) using the MapSplice informatics tool with default parameters
The mapped reads were further analyzed with Cufflinks to calculate the level of gene expression with FPKM
The levels of alternatively spliced isoforms were quantified with MISO (Mixture-of-Isoforms) probabilistic framework (Katz et al., 2010) using the annotated AS events for human hg19version 2.
The levels of alternatively spliced isoforms were also quantified with VAST-TOOLS using the event annotation as previously described
Genome_build: hg19
|
| |
|
| Submission date |
May 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Zefeng Wang |
| E-mail(s) |
wangzefeng@picb.ac.cn
|
| Organization name |
CAS-MPG Partner Institute for Computational Biology (PICB)
|
| Street address |
320 Yueyang Road
|
| City |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE81485 |
An extensive program of periodic alternative splicing linked to cell cycle progression |
|
| Relations |
| BioSample |
SAMN05004857 |
| SRA |
SRX1770019 |
