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Sample GSM2154760 Query DataSets for GSM2154760
Status Public on May 19, 2016
Title double thymidine block at 12h
Sample type SRA
 
Source name G1
Organism Homo sapiens
Characteristics cell cycle: G1
cell line: HeLa cells
sample type: double thymidine block at 12h
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 SAMN05004861
SRA SRX1770023

Supplementary file Size Download File type/resource
GSM2154760_D7.txt.gz 1.2 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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