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Sample GSM2123102

Query DataSets for GSM2123102

Status

Public on May 05, 2016

Title

H9 hESC 2

Sample type

SRA

Source name

H9 hESC

Organism

Homo sapiens

Characteristics

cell line: H9
cell type: human embryonic stem cells (hESCs)

Growth protocol

H9 human embryonic stem cell(hESCs) (WA09) from WiCell was cultured on a feeder layer of irradiated mouse embryonic fibroblasts following previously established protocols. H9 hESCs were detached from their feeder layer using 1mg/ml collagenase for 15-20 minutes and 0.5mg/ml dispase for an additional 15 minutes. Wells were washed with media to collect floating undifferentiated hESCs and colonies were dissociated using Accumax at 37°C for 10 minutes to generate a single cell suspension. At day 0, embryoid bodies were formed using the hanging drop method with 4500 cells/drop in DMEM/F12 media supplemented with 20% knockout serum replacement, 4ng/ml bFGF, NEAA and glutamine. After 2 days of hanging drop culture, embryoid bodies were transferred to sterile petri dishes with refreshed media. After 6 days in culture, embryoid bodies were transferred to new petri dishes containing neural induction media consisting of DMEM/F12, 1:100 N2 supplement, NEAA, glutamine and 1ug/ml heparin until day 11. At day 11, organoids were transferred to Matrigel droplets and cultured in 1:1 mixture of DMEM/F12 and Neurobasal medium supplemented with 1:100 B27 without vitamin A, 1:200 N2, NEAA, insulin, beta-mercaptoethanol and glutamine. Organoids were then transferred to stir flask bioreactors for long term growth on day 15 in the same differentiation media except with the addition of retinoic acid and vitamin A. Media was changed every 3 days.

Extracted molecule

total RNA

Extraction protocol

Total RNAs were collected using Qiagen RNeasy MiniKit following manufacturer's instructions
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Group 1
2_S2_R1_001.sorted

Data processing

BWA alignment to hg19
mRNA quantification and annotatino Ensembl in Partek Genomic Suite 6.6
Filtering RPKM for all samples <=0.5, data transformation (log2+1), normalization (quantile normalization)
Differential expression analysis (ANOVA) abs>=2, FDR adjusted P-value<=0.05
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample, fold change, p-value, etc

Submission date

Apr 14, 2016

Last update date

May 15, 2019

Contact name

Tariq Rana

E-mail(s)

trana@ucsd.edu

Organization name

University of California, San Diego

Department

Pediatrics