|
| Status |
Public on Nov 14, 2017 |
| Title |
hESC_day13_with_AA147_RNAseq_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Human embyonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Human Embryonic Stem Cell Line - HUES9
passages: P44-46
sample collection day: 13
agent: 10 microM AA147 (ATF6 Activator)
|
| Treatment protocol |
HUES9 Cells were differentiated performing spin embryoid body (EB) formation, cells were treated with 5 mM StemoleculeTM (Stemgent) 24 hrs prior to the procedure. Accutase (Innovative Cell Technologies, Inc.) was used to detach cells before performing cell counting using a hemocytometer. 10,000 cells were plated into each well of a 96 V-shaped plate (VWR) in the absence of FGF2 using differentiation media (KnockoutTM DMEM (Life Technologies); 20% KnockoutTM Serum Replacement (Life Technologies); 1% MEM Non-Essential Amino Acids Solution (Life Technologies); 1% Penicillin –Streptomycin (Life Technologies); 1% GlutaMAXTM (Life Technologies); 55 μM 2-mercaptoethanol (Fisher Scientific). Plates were spun down at 950 x g for 5 min (Beckman, AllegraTM X- 22), before placing at 37C, 5% CO2 to encourage differentiation. Suspension cultures were prepared 24 hrs after EB preparation, and EBs were transferred into ultra low attachment 6-well microplates (Corning Inc.) for 5 days until EBs were plated on to Matrigel (Corning Inc.) coated dishes for further differentiation. Throughout differentiation, samples were collected at indicated time points. Differentiation media, containing required drug treatments or compounds was changed daily. ATF6 activating compound AA147 and its analogue RP22 was prepared in DMSO as stock solution of 10 mM, working aliquots were prepared and stored at -20C. The compound was used in differentiation media at final concentration of 10 μM. DMSO was used as drug solvent control, but was excluded from data presentation when AA147 and RP22 were used since no difference between RP22 and DMSO treated samples was detected.
|
| Growth protocol |
HUES9 were maintained on a feeder layer of Mito-C treated primary MEFs (EMD Millipore), with daily media changes of pre-warmed conditioned media supplemented with 6 ng/ml FGF2 (EMD Millipore). Conditioned media was prepared by incubating primary MEFS for 24hrs with hESC specific media (KnockoutTM DMEM (Life Technologies); 10% KnockoutTM Serum Replacement (Life Technologies); Human Plasmanate (Chapin Healthcare); 1% MEM Non-Essential Amino Acids Solution (Life Technologies); 1% Penicillin -Streptomycin (Life Technologies); 1% GlutaMAXTM (Life Technologies); 55 μM 2-mercaptoethanol (Fisher Scientific). hESCs were used between passages 36-46 for all experiments. hESCs were split every three days using accutase (Innovative Cell Technologies, Inc.), spun out at 200Åã x g for 5 min (Beckman, AllegraTM X-22) before re-plating on freshly prepared PMEF cells. Primary MEFs were plated on to 0.1% gelatine coated 10 cm dishes using PMEF media (DMEM (Corning Inc.; 10% FBS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples for RNA-Seq analysis were collected at day 1 and 13 treated with 10 μM RP22 or 10 μM AA47. Three independent experiments were performed. Cells were lysed and total RNA was collected (RNeasy mini kit, Qiagen)
Libraries were constructed using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina Casava1.8.2 software was used for basecalling.
Single end, 100 bp-long reads from RNA Seq experiments were aligned to the GRCh37.p13 human genome reference assembly using SeqMan NGen 11.2.1 or 12.2.0 (DNAStar Inc.) using default parameters.
The assembly data were imported into ArrayStar 12.2 with Qseq (DNAStar Inc.) to quantify the gene expression levels. The sequence counts were normalized to reads per kilobase per million (RPKM) after filtering out non-mRNA sequence features.
Genome_build: GRCh37.p13 human genome reference assembly
Supplementary_files_format_and_content: abundance measurements in RPKM, tab-delimited text
Supplementary_files_format_and_content: abundance measurements in raw counts, tab-delimited text
|
| |
|
| Submission date |
Nov 13, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Evan Thomas Powers |
| E-mail(s) |
[email protected]
|
| Organization name |
The Scripps Research Institute
|
| Department |
Chemistry
|
| Street address |
10550 North Torrey Pines Rd
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE106847 |
The Unfolded Protein Response Regulator, ATF6, Promotes Mesodermal Differentiation |
|
| Relations |
| BioSample |
SAMN08016963 |
| SRA |
SRX3387524 |
