|
|
GEO help: Mouse over screen elements for information. |
|
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 Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
|
|
|
|
|