|
| Status |
Public on Sep 06, 2022 |
| Title |
12Z_siCHD4siARID1A_rep2_RNA |
| Sample type |
SRA |
| |
|
| Source name |
12Z endometriotic epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: 12Z endometriotic epithelial cells
treatment/condition: siCHD4+siARID1A (CHD4/ARID1A co-knockdown by siRNA)
|
| Treatment protocol |
Cells were seeded in antibiotic-free media the day before siRNA transfection. 50 nM siRNA (Dharmacon, ON-TARGETplus) were transfected into cells using the Lipofectamine RNAiMAX (ThermoFisher Scientific) reagent, according to the manufacturer protocol, in OptiMEM (Gibco). Growth media was replaced 24 hours following transfection, without antibiotics. 48 hours after transfection, low serum (0.5% FBS) growth media was added with antibiotics. Cells were harvested 72 hours following siRNA transfection.
|
| Growth protocol |
Human 12Z endometriotic epithelial cells were cultured in growth media (10% FBS, 1% L-glutamine, 1% Penicillin-Streptomycin).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were purified for RNA using the Quick-RNA Miniprep Kit (Zymo Research).
Transcriptome libraries were prepared and sequenced by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA mRNA HyperPrep kit (v4.17) (Kapa Biosystems). RNA was sheared to 300-400 bp. Prior to PCR amplification, cDNA fragments were ligated to IDT for Illumina unique dual adapters (IDT DNA Inc). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies), QuantiFluor dsDNA System (Promega), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Individually indexed libraries were pooled and 50 bp, paired-end sequencing was performed on an Illumina NovaSeq 6000 sequencer using a 100-cycle sequencing kit (Illumina). Each library was sequenced to an average raw depth of 20-25 million reads. Base calling was done by Illumina RTA3 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
mRNA (replicate #2) extracted from siCHD4+siARID1A treated (CHD4/ARID1A co-knockdown) 12Z endometriotic epithelial cells.
|
| Data processing |
Raw reads were trimmed with Trim Galore! (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) followed by quality control analysis via FastQC (Andrews 2010) and MultiQC (Ewels, Magnusson et al. 2016).
Trimmed reads were aligned to GRCh38.p12 (hg38) assembly and indexed to GENCODE (v28) along with gene feature counting via STAR (Dobin, Davis et al. 2013).
Low count genes with less than 1 count per sample on average were filtered prior to count normalization and differential gene expression (DGE) analysis by DESeq2 with Empirical Bayes shrinkage for fold-change estimation (Love, Huber et al. 2014, Love, Anders et al. 2015).
Wald probabilities were corrected for multiple testing by independent hypothesis weighting (IHW) (Ignatiadis, Klaus et al. 2016) for downstream analyses.
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: Tabular STAR gene-level feature raw read counts and transcriptome-wide differential gene expression analysis results from DESeq2.
|
| |
|
| Submission date |
Dec 09, 2021 |
| Last update date |
Sep 06, 2022 |
| Contact name |
Ronald L. Chandler |
| E-mail(s) |
rlc@msu.edu
|
| Organization name |
Michigan State University
|
| Department |
Obstetrics, Gynecology and Reproductive Biology
|
| Street address |
3100-4 400 Monroe NW
|
| City |
Grand Rapids |
| State/province |
MI |
| ZIP/Postal code |
49503 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE190555 |
ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers [12Z_siCHD4_siARID1A_RNA] |
| GSE190557 |
ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers |
|
| Relations |
| BioSample |
SAMN23818617 |
| SRA |
SRX13364011 |
