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Sample GSM2734322

Query DataSets for GSM2734322

Status

Public on Jul 23, 2018

Title

E9.5_VPR_Pdx1-pos_1_25

Sample type

SRA

Source name

Ventral Pdx1-expression region

Organism

Mus musculus

Characteristics

embryonic day: E9.5
sorting: Pdx1-GFP+

Growth protocol

Undifferentiated NKX6.1-mCherry Mel-1 hESCs were maintained on mouse embryo fibroblast feeder layers in hESC media as previously described (Kennedy et al., 2007). For generation of NKX6.1+ pancreatic progenitors, a protocol described by (Nostro et al., 2015) was used. Differentiation was started when NKX6.1-mCherry Mel-1 hESCs reached 80% confluence. Briefly, after removal of hESC media, cultures were treated with RPMI media containing 100 ng/ml of hActivin A and 2 mM CHIR 99021 (Tocris) for 1 day (d0–d1). Cells were treated with RPMI containing 100 ng/ml of hActivin A and 5 ng/ml hbFGF for 2 days (d1–d3). At day 3 of differentiation, cultures were treated with stage 2 media consisting of serum-free differentiation (SFD) media 63 containing 50 ng/ml of hFGF10 and 3 ng/ml mWnt3a. At d6 of differentiation, the medium was changed to DMEM with 1% vol/vol B27 supplement (without vitamin A) (Life Technologies), ascorbic acid (50 mg/ml; Sigma), KAAD-cyclopamine (0.25 mM; Stemgent), all-trans RA (2 mM; Sigma), hNOGGIN (50 ng/ml), and hFGF10 (50 ng/ml) for 2 days, with the medium changed every day. At d8, the medium was changed to DMEM with 1% vol/vol B27 supplement (Life Technologies), ascorbic acid (50 mg/ml; Sigma), hNOGGIN (50 ng/ml), hEGF (50 ng/ml), nicotinamide (10 mM; Sigma) and hActivin A (3 ng/ml). All cytokines were purchased from R&D Systems unless otherwise stated. Cultures were maintained in a 5% CO2, 5% O2, 90% N2 environment.

Extracted molecule

total RNA

Extraction protocol

Bulk-cell RNA-seq (Sample 001-016)
For E10.5, E14.5 and E17.5 dorsal and ventral pancreases, a total of 5-7 x 10e4 sorted cells were used to isolate total RNA using the Rneasy Micro Kit (Qiagen, 74004). Poly(A) RNA was further purified with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490).
Libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420) according to the manufacturer's instructions.
Single-cell RNA-seq (Sample 023-541, 568-586, 607-631, 671-674)
Libraries were prepared according the Smart-seq2 method as previously reported (Picelli et al., 2014).
2 ng cDNA were subjected to prepare the libraries using the TruePrep DNA Library Prep Kit (TD502, Vazyme).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Sample 631
processed data file:
Single-cell_RNA-seq_Read_Count.txt.gz
Single-cell_RNA-seq_RPKM.txt.gz

Data processing

Bulk-cell RNA-seq (Sample 001-016)
Sequencing reads were aligned to the Mus musculus reference genome GRCm38/mm10 using TopHat (v2.1.0) with parameter "-o out_dir -G gtf --transcriptome-index trans_index bowtie2_index input_fastq1 input_fastq2".
Reads aligned to genes were counted by HTSeq (v 0.6.0) with parameters "-f bam -r pos -s reverse -a 30".
Reads of biological replicates were pooled.
Gene expression levels were quantified as reads per kilobase of longest transcript per million mapped reads (RPKM).
Single-cell RNA-seq (Sample 023-541, 568-586, 607-631, 671-674)
Sequencing reads were aligned to the Mus musculus reference genome GRCm38/mm10 using TopHat (v2.1.0) with parameter "-o out_dir -G gtf --transcriptome-index trans_index bowtie2_index input_fastq".
Reads aligned to genes were counted by HTSeq (v 0.6.0) with parameters "-f bam -r pos -s no -a 30".
Gene expression levels were quantified as reads per kilobase of longest transcript per million mapped reads (RPKM).
Genome_build: mm10
Supplementary_files_format_and_content: values tab-delimited text files include read count and RPKM values for each sample

Submission date

Aug 07, 2017

Last update date

May 15, 2019

Contact name

Cheng-ran Xu

Organization name

Peking University

Department

School of Basic Medical Sciences

Street address

NO.5 YIHEYUAN ROAD HAIDIAN DISTRICT, BEIJING, P.R.CHINA