|
| Status |
Public on May 21, 2019 |
| Title |
rnaseq_control2_DMSO |
| Sample type |
SRA |
| |
|
| Source name |
dermal fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
passage: 1
disease state: healthy control
treatment: DMSO
cell type: dermal fibroblasts
|
| Treatment protocol |
Control and SSc fibroblasts were treated with DMSO or JQ1 (0.25 uM) for 48 hours in complete cell culture media.
|
| Growth protocol |
Primary dermal fibroblasts lines were established from skin biopsies taken from healthy control donors or patients with diffuse cutaneous SSc. Fibroblasts were cultured at 70-80% confluency in DMEM + 10% FCS + anti/anti + glutamax, and subjected to ATAC-seq and RNA-seq. For treatment studies, fibroblasts were treated with DMSO or JQ1 prior to RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Chromatin lysates were isolated upon centrifugation after cell-lysis. RNA was isolated by trizol and RNA isolation columns.
ATAC-seq: Nuclei were isolated from three control and three SSc fibroblast lines using ATAC lysis buffer, transposed with Tn5 transposase (Illumina) for 30 minutes, and ligated with barcoded adapter sequences as previously described (Buenrostro JD et al. 2013. Nature. 10:1213-8). Library quantitation and quality check was performed using an Agilent 2100 Bioanalyzer prior to sequencing as previously described, and 50 base-pair paired-end FASTQ reads were generated by Illumina HiSeq 2000. RNA-seq: RNA was isolated from three control and three SSc fibroblast lines using Trizol and RNeasy isolation kit (Qiagen). DNA was digest with on-column DNase treatment (Qiagen). The quality of the RNA used for sequencing was determined using an Agilent 2100 Bioanalyzer; all samples had RNA integrin numbers (RIN) of at least 9.60. mRNA was enriched by poly-A selection, prepped using an Illumina TruSeq mRNA sample preparation kit, and sequenced by Illumina HiSeq 2000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
normalized_counts_rnaseq_dmsojq1_MERGE.txt
|
| Data processing |
ATAC-seq: basecalling was performed using CASAVA. Reads were trimmed for adapter sequences, and aligned to UCSC hg19 using bowtie2. Duplicate fragments were identified and removed using PICARD tools. Reads were filtered for alignment quality greater than Q30. Reads that mapped to mitochondria, unmapped contigs, or the Y chromosome were removed. Sequence reads were subsequently filtered for nucleosome-free reads based on read fragment size limits (0 to 100 bp), which were determined previously (Buenrostro JD et al. 2013. Nature. 10:1213-8). Peaks were called using MACS2
RNA-seq: bascalling was performed using CASAVA. RSEM was used to align reads to UCSC hg19 and to generate gene expression levels. Count output from RSEM was used as input for DESeq2 differential gene expression analysis.
Genome_build: hg19
Supplementary_files_format_and_content: ATAC-seq: bigwig files. RNA-seq (first set): tab-delimited text containing normalized read couts.
|
| |
|
| Submission date |
Apr 25, 2019 |
| Last update date |
May 21, 2019 |
| Contact name |
Joseph Y Shin |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Street address |
733 N Broadway MRB-539
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE130313 |
Epigenetic activation and memory at a TGFB2 enhancer in systemic sclerosis |
|
| Relations |
| BioSample |
SAMN11506833 |
| SRA |
SRX5735741 |
