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Sample GSM5963957 Query DataSets for GSM5963957
Status Public on Sep 16, 2022
Title F47-NoE2-S1_C
Sample type SRA
 
Source name Meniscal Fibrochondrocytes
Organism Homo sapiens
Characteristics tissue: Meniscus
cell type: Meniscal Fibrochondrocytes
experiment: Experiment 1
age: 47-year-old
gender: Female
treatment: Control (No E2)
treatment duration: 72 h
flow cell: C
Treatment protocol Cells were treated in triplicate with 17β-estradiol (E2, Sigma, E2758) in pfDMEM supplemented with 15% charcoal/dextran treated FBS and 1% penicillin/streptomycin under pulsed or continuous dosing. For pulsed dosing, cells were exposed to E2 for 1 h, after which the E2 media was replaced with E2-free media for 23 h. This pattern was repeated for the length of the experiment. For continuous dosing, cells were exposed to E2 for the entire treatment period.
Growth protocol Cells were cultured in tissue culture flasks in DMEM supplemented with 10% Premium FBS (Atlanta Biologicals, 511150) and 1% penicillin/streptomycin and passaged at 70-80% confluency using trypsin (Sigma, T4174, diluted to 1x before use). When passage 1 cells reached 70-80% confluency, cells were washed with PBS (Gibco, 20012-207) and cultured in phenol red-free DMEM (pfDMEM, Gibco, 31053-028) with 15% charcoal/dextran treated FBS (Atlanta Biologicals, S11650) for 3 days to starve them of endogenous hormones. Cells were then passaged and seeded in 24 well plates in pfDMEM and allowed to adhere overnight.
Extracted molecule total RNA
Extraction protocol For Experiment 1 cells were collected in TRIzol, and total RNA was purified using a PureLink RNA Mini Kit (Life Technologies, 12183025) according to the manual. DNA was removed using a DNA-free DNA Removal Kit (Life Technologies, AM1906) according to the manual. For Experiment 2 cells were collected and total RNA was purified using a QIAGEN RNeasy Mini Kit (QIAGEN, 74104) with no DNase treatment.
Sequencing libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760). The sequencing library construction process included mRNA purification with polyA selection beads, fragmentation to inserts of ~200 bp, strand specific cDNA synthesis, end repair, 3’ end adenylation, adapter ligation, and PCR amplification. Samples were run across 3 flow cells (Experiment 1: A, B, and C; Experiment 2: 708, 709, and 710).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model NextSeq 550
 
Data processing Base calling was carried out by the Illumina NextSeq 550 Real Time Analysis (RTA) software.
Raw reads were filtered using fastp (Experiment 1) or trimmomatic 0.39 (Experiment 2) and aligned to the reference transcriptome using Kallisto 0.46.1-1.
Kallisto 0.46.1-1 was used for transcript abundance estimates
tximport was used to import Kallisto transcript abundance estimates into DeSeq2 1.3 for differential expression analysis
Assembly: ENSEMBL Homo sapiens reference transcriptome build GRCh38 release 101
Supplementary files format and content: .tsv files containing raw quantified counts and TPM counts
 
Submission date Mar 21, 2022
Last update date Sep 16, 2022
Contact name Jennifer L Robinson
E-mail(s) jlrobinson@ku.edu
Organization name University of Kansas
Department Department of Chemical and Petroleum Engineering
Street address 1530 W. 15th St
City Lawrence
State/province Kansas
ZIP/Postal code 66045
Country USA
 
Platform ID GPL21697
Series (1)
GSE199087 RNA sequencing of human meniscal fibrochondrocytes treated with 17beta-estradiol
Relations
BioSample SAMN26852691
SRA SRX14534888

Supplementary file Size Download File type/resource
GSM5963957_t_F47-NoE2-S1_S1_R1_C.tsv.gz 2.3 Mb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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