|
| Status |
Public on May 30, 2018 |
| Title |
RNA-Seq P4-EBV EV |
| Sample type |
SRA |
| |
|
| Source name |
EBV (Patient 4)_empty vector
|
| Organism |
Homo sapiens |
| Characteristics |
patient diagnosis: hyper-IgE syndrome (HIES)
cell type: EBV-B
antibodies: n/a
|
| Treatment protocol |
EBV-B cells were transduced with ZNF341 WT isoforms (iso 1 or iso 2) or empty vectors. TCR stimulation: Fresh isolated CD3+ T cells were activated by incubation for 24 or 48 hours with antibodies against CD3 and CD28.
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density centrifugation (Amersham-Pharmacia-Biotech) from cytopheresis or whole-blood samples obtained from healthy volunteers and patients, respectively. PBMCs, PHA blasts and Epstein Barr virus-immortalized lymphoblastoid cell lines (EBV-B cells) were cultured in RPMI-1640 medium supplemented with 10% FCS (referred to hereafter as complete medium).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-Seq, total RNA extracted using the RNeasy Mini Kit (Qiagen). Poly(A)+ RNA was selected twice from total RNA using Dynal oligo magnetic beads (Invitrogen). Double-stranded cDNA was synthesized using SuperScript (Invitrogen) and fragmented using Bioraptor (Diagenode). Adaptors (Illumina) were ligated to the dsDNA using T4 DNA ligase (New England Biolabs) after end-repair using End-It kit (Epicentre) and dA addition. Libraries were size-selected for 250-400 bp fragments, purified using 2% E-Gel (Invitrogen), and amplified for 18 cycles by PCR with PE 1.0 and PE 2.0 primers (Illumina) and Phusion High Fidelity PCR Master Mix. For ChIP-Seq, chromatin was isolated from 10 million cells, after cross-linking with 1% methanol-free formaldehyde (Thermo Fisher Scientific). Chromatin was fragmented by sonication and subjected to immunoprecipitation with 10 µg of either mouse IgG (Santa Cruz Biotechnology, Dallas TX) or mouse monoclonal anti-ZNF341 (8B3.1) antibodies prebound to Magna ChIPTM Protein A+G Magnetic Beads (Millipore, Billerica MA). The immunoprecipitate was washed and cross-linked, and ChIP-Seq DNA libraries were then prepared with the fragmented DNA and the KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNA-Seq P4-EBV Empty Vector
|
| Data processing |
Basecalls performed using CASAVA 1.8. Raw reads were aligned to the human hg19 genome assembly using Bowtie 0.12.9 or Tophat 2.0.11
Aligned reads are converted to bam format using samtools and bedGraph files are converted using bedtools. ChIP-Seq peaks are called using Model-based Analysis of ChIP-Seq (MACS) version 1.4.2. RPKM (reads per kilobase per million reads) was calculated using unpublished python script to normalize gene expression values and R package edgeR is used to identify differentially expressed genes. Browser-extensible data files are converted to binary tiled data files (TDFs) with IGVTools 2.3.32 for browser viewing.
Genome_build: hg19
Supplementary_files_format_and_content: text file with RPKM for RNA-seq samples, bedGraph files with genome-wide coverage data (20-bp window) for ChIP-seq.
|
| |
|
| Submission date |
Apr 16, 2018 |
| Last update date |
May 30, 2018 |
| Contact name |
Peng Li |
| E-mail(s) |
peng.li@nih.gov
|
| Organization name |
NIH
|
| Department |
NHLBI
|
| Lab |
LMI
|
| Street address |
9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE113194 |
A recessive form of hyper-IgE syndrome by disruption of ZNF341-dependent STAT3 transcription and activity |
|
| Relations |
| BioSample |
SAMN08937982 |
| SRA |
SRX3942985 |
