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Sample GSM2047268

Query DataSets for GSM2047268

Status

Public on Aug 15, 2017

Title

SEN hADSC

Sample type

SRA

Source name

adipose derived mesenchymal stem cells

Organism

Homo sapiens

Characteristics

cell type: adipose derived mesenchymal stem cells (hADSCs)
cell subtype: Senescent

Growth protocol

Human adipose derived stem cells were isolated from human subcutaneous white adipose tissue collected during liposuction procedures. The lipoaspirate was suspended in Hank’s Buffered Salt Solution (HBSS, Life technology), 3.5% Bovine Serum Albumin (BSA, Sigma), 1% Collagenase Type II (Sigma) in 1:3 w/v ratio and shaken at 37°C for 50 min. The cells were filtered through a 70 μm mesh cell strainer (BD Falcon #352350), treated with Red Blood Cell Lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3), and expanded ex vivo in DMEM/F12 complete medium (DMEM/F12, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin; Life technology) in 10% CO2 at 37°C and passaged at 80% confluency, changing medium every 72–96 h. Cumulative population doublings were calculated by summing the population doublings (PD = log(N/N0) x 3.33, where N0 is the number of cells plated in the flask and N is the number of cells harvested at this passage) across multiple passages as a function of the number of days it was grown in culture. Five x 105 cells each were incubated for 30 min on ice in the dark with fluorochrome-conjugated antibodies (CD31, CD44, CD45 and CD105; Invitrogen) in PBS with 1% BSA (Sigma), washed and analyzed in a Guava EasyCyte Mini System (Guava Technologies, Millipore). Data analysis was done with FlowJo software (Tree Star, Ashland, OR). A senescence-associated β-galactosidase activity assay was done according to manufacturer’s instructions (BioVision). The cells were washed with PBS and fixed with fixation solution for 15 min at room temperature. The cells were washed with PBS twice and X-gal staining solution was added with a staining supplement per well and incubated overnight at 37°C. The cells were washed twice with PBS, and the images were captured using a microscope (Nikons, TE300, DXM1200 Digital Camera, Japan).

Extracted molecule

total RNA

Extraction protocol

Cells were isolated from human subcutaneous white adipose tissue collected during liposuction procedures. Total RNA was extracted to construct RNA-seq library.
Total RNA was isolated from 3 different genetic lines of adipose derived stem cells in SR and SEN conditions. Samples were grouped together for SR and SEN. Total RNA preparation has been processed with RiboMinus kit to significantly reduce ribosomal RNA in the sample. All samples have been precipitated with Pellet-Paint and dissolved in depc-treated millipure water. SuperScript™ ds cDNA synthesis was performed with random-primers. GS FLX Titanium General Library Preparation Method was employed to incorporate Multiplex Identifier (MID)-containing adaptors for General (e.g.Shotgun) library preparation. Unidirectional Sequencing of Amplicon Libraries Using GS FLX Titanium emPCR Kits (Lib-L) according to Roche Protocol APP No.001-2009 GS FLX Titanium MID-1 ExST Primer1 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGACGAGTGCGTGAGCGGCCGCGAAGATCAGA-3’ GS FLX Titanium MID-3 ExST Primer1 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGAGACGCACTCGAGCGGCCGCGAAGATCAGA-3'

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

454 GS FLX Titanium

Description

processed data file: SR_SEN_RNA-seq_gene_read_counts.xlsx
processed data file: SR_SEN_RNA-seq_normalized_gene_expression_RPKM.xlsx

Data processing

Individual sequence reads were mapped to the human genome reference sequence (UCSC hg18; NCBI build 36.1) using the program BLAT.
BLAT was run with default settings with the exception that the minimal sequence identity was set to 99%
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Mortazavi et al, Nature, 2008. The number of reads falling in the exons of each transcript were counted and normalized by the size of the total exome length and by the size of the library size.
Genome_build: hg18
Supplementary_files_format_and_content: Excel files include read counts and RPKM (normalized abundance) values for both samples

Submission date

Jan 27, 2016

Last update date

May 15, 2019

Contact name

Victoria Lunyak

E-mail(s)

vlunyak@aelanct.com

Phone

415-488-6041

Organization name

Aelan Cell Technologies