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Sample GSM2212218

Query DataSets for GSM2212218

Status

Public on Sep 12, 2016

Title

MCF10controlcell

Sample type

SRA

Source name

MCF10A cells

Organism

Homo sapiens

Characteristics

cell line: MCF10A
cell type: normal mammary epithelial

Growth protocol

The human mammary epithelial cell line MCF10A, and breast cancer cell lines MCF7 and MDA-MB-231 were obtained from the American Type Culture Collection (Manassas, VA). MCF10A cells were cultured in DMEM/F12 medium supplemented with 5% exosome-depleted horse serum, 20ng/ml epithelial growth factor, 0.5mg/ml hydrocortisone, 100ng/ml cholera toxin, 10 μg/ml insulin, 100 IU/mL penicillin and 100 μg/mL streptomycin. MCF7 and MDA-MB-231 cells were cultivated in DMEM medium supplemented with 10% exosome-depleted fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin (Corning/Mediatech, Inc. Manassas, VA). Exosome-depleted FBS and horse serum were prepared by pelleting the serum exosomes by ultracentrifugation at 100,000 x g for 2 h at 4°C, and the resulting supernatant was filtered through a 0.2 μm pore filter. Cells were routinely maintained in a humidified chamber at 37°C and 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Exosomes were isolated utilizing a combination of centrifugation, ultracentrifugation, and filtration as we have previously described [16] or with the Exoquick-TC reagent (System Biosciences, Mountain View, CA) following the manufacturer’s protocol. For ultracentrifugation isolation, conditioned cell culture media was collected and centrifuged at 10,000 x g for 30 min at 4°C, to remove cells and large debris. The supernatant was filtered using a 0.22-μm pore filter and the exosomes were pelleted at 100,000 x g for 1 h at 4°C. The exosome pellet was washed with 10 ml of 1X PBS and pelleted again by centrifugation at 100,000 x g for 1 h at 4°C. The resulting pellet was either suspended in 1X PBS for whole exosome applications or further processed for RNA or protein extraction. Total RNA was extracted from exosome pellet using the TRIzol reagent (Invitrogen/Life Technologies) following the manufacturer’s protocol. RNA concentration was quantitated using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Small RNA libraries were constructed using New England Biolabs (NEB) NEBNext Multiplex Small RNA Library Prep Set for Illumina sequencers and the NEB standard protocol. Individual libraries were constructed using 1µg of total RNA isolated from each sample.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina MiSeq

Data processing

Each library was indexed in order to multiplex four samples per sequencing run on the Illumina MiSeq platform using MiSeq 50 cycle Reagent Kits v2. A minimum of 17 million 50bp sequencing reads were collected from each sample and data were analyzed using Genesifter software (formerly Geospiza) (PerkinElmer, Santa Clara CA). Raw data for each sample were aligned to the most recent mirBASE database (mirBase.org [17]) with remaining reads aligned to the most recent human genome (hg18 build [18]) build in order to identify previously unknown regions that may encode for unique miRNAs. Pairwise comparison of the alignment results was done using Genesifter for identification of miRNAs that are differentially expressed at a significant level, i.e. up- or down-regulated.

Submission date

Jun 23, 2016

Last update date

May 15, 2019

Contact name

Bethany Hannafon

E-mail(s)

bethany-hannafon@ouhsc.edu

Organization name

University of Oklahoma Health Sciences Center

Department

Obstetrics and Gynecology