|
| Status |
Public on Feb 21, 2023 |
| Title |
Adult chondrocytes2_STAT3 |
| Sample type |
SRA |
| |
|
| Source name |
Chondrocytes isolated from adult tissue samples
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: STAT3 antibody
cell type: chondrocytes
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
In situ chromatin profiling using CUT & RUN was performed according to Skene et al 34. Briefly samples were FACS sorted using DAPI to select live cells and 10,000 cells were collected in 10%FBS-PBS media. Cell nuclei were immobilized on Concanavalin A beads after washing. pSTAT3 or normal rabbit IgG antibodies (Cell signaling technology) were incubated with the nuclei overnight in the presence of 0.02% digitonin at 4°. The next day, 700ng/mL of proteinA-micrococcal nuclease (pA-Mnase purified in house with vector from Addgene 86973 76) were incubated with the nuclei at 4 degrees for an hour. After washing, the tubes were placed in heat blocks on ice set to 0 degrees, CaCl2 (1mM) was added and incubated for 30 min before 2X Stop buffer containing EDTA was added. DNA was extracted using Qiagen DNA isolation kit according to manufacturer’s protocol Purified DNA was quantified in Qubit and Bioanalyzer (2100) traces using D5000 high sensitivity chip were run to determine the size of the cleaved products. UMI-coded libraries were generated using Swift Biosciences-ACCEL-NGS® 2S PLUS DNA LIBRARY KITS according to manufacturer’s protocol. Pair-end (75bp) Illumina sequencing was performed on the UMI-coded and amplified libraries using NextSeq platform.
Swift Biosciences-ACCEL-NGS® 2S PLUS DNA LIBRARY KITS
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: Cut&Run
UMI-tools ‘extract’ function was used to remove UMIs from each read of the raw fastq files and append them to the read name. The initial quality of the raw fastq files were checked using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were trimmed using Cutadapt v2.10 in paired-end mode. Trimmed reads were aligned to human genome build hg19 using bowtie2. Next, aligned reads were deduplicated and PCR duplicates were removed by UMI-tools ‘dedup’ function followed by sorting and indexing of the deduplicated bam files by SAMtools v1.11. Bam coverage maps were generated using bamCoverage from the deepTools suite v3.5.0. Heatmaps were generated using computeMatrix and plotHeatmap from the deepTools suite v3.5.0. Significant peaks (p-value<0.05) were called from the deduplicated reads using MACS2 and annotated using the R package ChIPseeker.
Genome_build: hg19
Supplementary_files_format_and_content: MACS2 peak files
|
| |
|
| Submission date |
Aug 02, 2021 |
| Last update date |
Feb 21, 2023 |
| Contact name |
Denis Evseenko |
| E-mail(s) |
evseenko@usc.edu
|
| Phone |
323-219-0234
|
| Organization name |
University of Southern California
|
| Department |
Orthopaedic Surgery
|
| Lab |
Evseenko Lab
|
| Street address |
1450 Biggy St
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE181351 |
Identification of the key epigenetic determinants of chondrocyte aging in humans (Cut&Run) |
| GSE181356 |
Identification of the key epigenetic determinants of chondrocyte aging in humans |
|
| Relations |
| BioSample |
SAMN20523550 |
| SRA |
SRX11632672 |
