 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 01, 2015 |
| Title |
HD291 line after 15 mechanic passages |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cell line
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
cell line: HD291
cell type: embryonic stem cells
passage: p13+MP15
|
| Treatment protocol |
Mechanical passaging was carried out under an inverted microscope under the hood using scalpels (Assou et al., 2009; Bai et al., 2011). For enzymatic single cell passaging, the cells were pre-treated with Y-27632 for 1h and dissociated with TrypLE™ Select (Invitrogen) for 10 mn at 37ºC. The reaction was stopped by hESC culture media and single hESC were released by pipetting and passed through a 20 µm strainer to eliminate hFF feeders, then counted and seeded on newly prepared feeder in density of 20 000 cells/cm2 at beginning and progressively to 3 000 cells/cm2 when cells were adaptated to enzymatic passaging. Both mechanic and enzymatic passaging were performed weekly. Experiments on HD291, HD129 and HS306 lines were initiated at p13, p16 and p25 respectively (termed initiating passages) and maintained independently using mechanic passaging (+MP) or enzyamatic passaging (+EP). All cell cultures were regularly checked for mycoplasma contamination and remained negative throughout the experiments.
|
| Growth protocol |
These lines were maintained in culture media consisting of 80% KO-DMEM, 20% KOSR, 2 mM L-glutamine, 1% non-essential amino acids, 0.5 mM β-mercaptoethanol (all from Gibco Invitrogen, Cergy-Pontoise, France) and complemented with 10 ng/mL bFGF (Abcys, Paris, France). These cells were maintained in 35-mm dishes with pre-coated irradiated (40 Gy) human foreskin fibroblast (hFF) feeders and were either enzymatically or mechanically passaged every week.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
In mechanically passaged culture, the hESC colonies were exscinded carefully using a scalpel. In enzymatic-passaged culture, the singles cells were released as described above. As SSEA4 is one of most obstinate pluripotence markers (Ramirez et al., 2011), hESC cells were purified and enriched using the magnetic beads Dynabeads® SSEA-4 (Invitrogen) and the manufacture’s instructions were rigorously followed. The quality control of the purification showed high efficiency and no over-selection of SSEA4+ sub-population. Total RNA was purified from cell culture using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) following manufacture’s instructions. We added an RNAse-Free DNase step in RNA extraction and an RNsae A treatment in DNA extraction to eliminate residual DNA or RNA.
QC of the quantity of the total RNA using nanodrop. Library construction using TruSeq RNA Sample Prep Kits. Library Quality Control and quantification by Bioanalyzer Agilent 2100, qPCR and Picogreen.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Transcriptome human embryonic cell line HD291 after 15 mechanic passages + initial passages 13 mechanic passages
|
| Data processing |
TopHat (http://tophat.cbcb.umd.edu)/Cuffinks(http://cufflinks.cbcb.umd.edu) was used in order to map reads to the refence genome according to annotated trasncripts.
The coverage depth of each unique exon was computed from the Tophat alignement. From the alignment generated by TopHat, the program htseq-count (part of the DESeq software package developed by Simon Anders (2010)) was used to retrieve the counts of reads which aligned to each transcript's exon. The exon annotations are based on Ensembl reference GTF 63. For each unique exon, the count information is reported for all related transcripts. If 2 transcripts contain one common exon, the count information for this exon is the same for both transcripts.
The program Cuffdiff (part of the Cufflinks software package) was used to compute the abundance of transcripts in each sample. From the Tophat mapping results it eval- uates the abundance of each Ensembl annotated transcript.
The metric used by Cufflinks to estimate the abundance of transcripts is the FPKM: Fragments Per Kilobase of exon per Million fragments mapped. The FPKM was introduced by Trapnell C et al. (2010). The Fraction of Major Isoform (FMI) indicate the relative proportion of each isoform for gene having multiple transcripts. It is equal to 100 for transcripts having the highest FPKM.
Genome_build: GRCh37.63
Supplementary_files_format_and_content: text files containing calculated FPKM and FMI for each annotated gene and transcript (Ensembl).
|
| |
|
| Submission date |
Jun 14, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Qiang BAI |
| E-mail(s) |
qiang.bai@uliege.be
|
| Phone |
+32 (0)4 366 9841
|
| Organization name |
University of Liege
|
| Department |
GIGA Institute
|
| Lab |
Immunophysiology Laboratory
|
| Street address |
Avenue de l'Hopital 1
|
| City |
Liège |
| ZIP/Postal code |
4000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE47917 |
Genome alterations in human pluripotent stem cells as very early events highly dependent on cell passaging conditions |
| GSE47946 |
Transcriptome of embryonic stem cells cultured in enzymatic and mechanic passaging methods [RNA-seq] |
|
| Relations |
| BioSample |
SAMN02203917 |
| SRA |
SRX305874 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1163071_HD291_p13+MP15_FPKM_FMI_values_comparisons.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
