|
| Status |
Public on Oct 26, 2023 |
| Title |
PolyA_SBNO2KD NHDF_TNF6h_h6 |
| Sample type |
SRA |
| |
|
| Source name |
Human normal dermal fibroblast
|
| Organism |
Homo sapiens |
| Characteristics |
gene manipulation: siSBNO2#3
stimulation: 10ng /ml TNF 6h
subcellular fractionation: Total
cell type: normal dermal fibroblast
|
| Treatment protocol |
For PAR-CLIP, FH-SBNO2 were induced by 1 μg/ml tetracycline for 24 hours before cross-linking in the 70% confluent dishes. 100 mM of 4 thiouridine (4-SU) was added to the culture medium 12 hours before cross-linking. For Poly A selected RNA-sequencing of SBNO2 KD fibroblasts, 0.2 million of NHFD from three different healthy neonatal donors (Lonza) were transfected with siSBNO2 (Thermo, HSS177015) as described above. After 72 hours, the cells were left without stimulation or stimulated with 10 ng/ml recombinant human TNF-a (PeproTech) for 1 hour or 6 hours.
|
| Growth protocol |
HEK293 and human normal dermal fibroblasts were cultured in DMEM supplied with 10% FBS and penicillinG/Streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For PAR-CLIP, Cells were cross-linked with 5000 uJ/cm2 of UV light (312nm) and fractionated nuclei were lysed and ribonucleocomplex were immunoprecipitated with anti-FLAGAb. Fluorescently labeled adaptors were ligated to the RNA and separated by SDS-PAGE. cDNA were synthesized by reverse transcript and subjected to library prep.
For PAR-CLIP, the cDNA was amplified by PCR and libraries were size selected by Pippinprep. For RNA seq, rRNA depletion was performed with the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (New England BioLabs) and sequencing library construction was performed with NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads (New England BioLabs) according to the manufacturer’s instructions. Each sample was multiplexed with NEBNext® Multiplex Oligos for Illumina® (96 Index Primers) (New England BioLabs). For poly A plus RNA-Seq from human fibroblasts, RNA was extracted using RNeasy Plus Mini Kit (Qiagen). Purified RNA was poly (A) enriched and sequenced by Novogene Co.Ltd (California, USA).
PAR-CLIP-seq, RNA-seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
NC_T6-SB_T6_gene_exp.diff
|
| Data processing |
Base calling and demultiplexing was performed using bcl2fastq/2.20.0
For RNA-Seq fastq files were aligned to human genome (hg38) using STAR aligner star/2.7.2b.
Differential expression was performed using cuffdiff (cufflinks/2.2.1)
PAR-CLIP reads where analyzed using PARpipes including: removal of PCR duplicates, adapter trimming (cutadapt 2.4 with Python 3.7.2 ), alignment to hg38 using bowtie/1.2.2 binding sites, cluster identification paralyzer
Genome_build: hg38
Supplementary_files_format_and_content: PAR-CLIP: comma separated values (clusters.csv) RNA-Seq: tab separated values .diff files
|
| |
|
| Submission date |
Nov 03, 2021 |
| Last update date |
Oct 26, 2023 |
| Contact name |
Markus Hafner |
| E-mail(s) |
markus.hafner@nih.gov
|
| Organization name |
NIH
|
| Department |
NIAMS
|
| Lab |
Laboratory of Muscle Stem Cells and Gene Regulation
|
| Street address |
50 South Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL21290 |
| Series (1) |
| GSE187784 |
Genome-wide maps of SBNO2 target transcripts and RNA-seq of SBNO2 deficient cells |
|
| Relations |
| BioSample |
SAMN22869467 |
| SRA |
SRX12966725 |
