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Sample GSM1093231

Query DataSets for GSM1093231

Status

Public on Jun 01, 2014

Title

DE2

Sample type

SRA

Source name

definitive endoderm cells

Organism

Homo sapiens

Characteristics

cell type: definitive endoderm cells

Treatment protocol

Primary human islet were obtained from Integrated Islet Distribution Program (IIDP, study number BS208) and processed according to a previous report with minor modification (Dorrell et al., 2008; Dorrell et al., 2011). The following antibodies were used to label desired cell populations: HPi2 (mouse IgG1), and HPa1 (mouse IgM) as primary antibodies (kindly provided by Dr. Streeter), Alexa647-conjugated donkey anti-mouse IgG and Alexa488-conjugated donkey anti-mouse IgM as secondary antibodies. Beta cells were recognized as HPi+/HPa-, and HPi+/HPa+ for alpha cells as described (Dorrell et al., 2011).

Growth protocol

Human ESC line H9 was used to differentiate into definitive endoderm cells and pancreatic progenitors as previously described (Jiang et al., 2011). The following antibodies (all were purchased from BD) were used to label different cell populations: PE-conjugated mouse anti-human SSEA4 and APC-conjugated mouse anti-human CD184 for undifferentiated human ESCs (SSEA4+/CD184-); APC-conjugated mouse anti-human CD184 IgG and PE-conjugated mouse anti-human CD117 for definitive endoderm (CD184+/CD117+); PE-conjugated mouse anti-human CD24 and FITC-conjugated rat anti-human CD49f for pancreatic progenitors (CD24high/CD49flow).

Extracted molecule

total RNA

Extraction protocol

Total RNA was purified from 1,000 sorted cells using ZR RNA microprep kit (Zymo Research, UAS). The cDNA synthesis and amplification was performed with the SMARTer ultra-low input RNA kit (Clontech, US).
The amplified cDNA (10-40 ng) was then fragmented by Covaris S2 sonicator (Covaris, US) and converted to sequencing libraries following the Illumina’s construction protocol for low input DNA (Illumina, US). Barcoded libraries were pooled and sequenced in Illumina Hiseq 2000 instrument (pair-end 100bp).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

processed data file for protein-coding genes:
processed_FPKM_genes_human_pancreatic_lineage.txt
processed data file for lncRNAs:
processed_FPKM_lncRNAs_human_pancreatic_lineage.txt

Data processing

All obtained reads from each sample were mapped against the human genome (hg19 build) with Bowtie/Tophat v2.0.2 which allows mapping across splice sites by reads segmentation (Trapnell et al., 2012). All programs were performed with default setting (unless otherwise specified).
The unique mapped reads (69-91% of total reads) were subsequently assembled into transcripts guided by reference annotation (Gencode v14 and Refseq gene models) with Cufflinks v2.0.2 (Trapnell et al., 2012). Expression level of each gene was quantified with normalized FPKM (fragments per kilobase of exon per million mapped fragments).
For the analyses of lncRNAs, cufflinks was used to estimate the transcript level FPKM values with the Gencode v14 lincRNA annotation (Derrien et al., 2012), and human body map lincRNA annotations (Cabili et al., 2011).
processed data file for protein-coding genes:
processed_FPKM_genes_human_pancreatic_lineage_140112.txt
processed data file for lncRNAs:
processed_FPKM_lncRNAs_human_pancreatic_lineage_140112.txt
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample.

Submission date

Mar 05, 2013

Last update date

May 15, 2019

Contact name

Yuting Liu

E-mail(s)

ytliu1985@gmail.com

Organization name

Harvard Medical School

Department

Department of Genetics