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Sample GSM4211342 Query DataSets for GSM4211342
Status Public on Aug 07, 2020
Title ADSCs-2
Sample type SRA
Source name Adipose-derived stem cells
Organism Homo sapiens
Characteristics passages: 6-7
donor's age: 24
biopsy site: thigh
Growth protocol Cells were cultured in complete culture medium for adult ADSCs ( Cyagen,CHN)
Extracted molecule total RNA
Extraction protocol Cultured cells were harvested with trypsin digestion.The total RNA was extracted and tested to be qualified.mRNA were enriched using poly-T oligo-attached magnetic beads.Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100system.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq platform and 150 bp paired-end reads were generated.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Data processing Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. We selected TopHat as the mapping tool for that TopHat can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels (Trapnell, Cole, et al., 2010).
EdgeR package(Version 3.3.3) was used for the differential expression analysis.P-value of 0.05 and log2(Fold change) of 1 were set as the threshold for significantly differential expression.
Genome_build: human NCBI genome build 36
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...
Submission date Dec 09, 2019
Last update date Aug 07, 2020
Contact name Fangfei Nie
Organization name Peking university third hospital
Department Department of Plastic Surgery
Street address 49 North Garden Rd.,Haidian District
City Beijing
ZIP/Postal code 100191
Country China
Platform ID GPL24676
Series (1)
GSE141708 mRNA profiles of human Dedifferentiated Adipose Cells and Adipose-derived stem cells in young donors
BioSample SAMN13516235
SRA SRX7293427

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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