|
| Status |
Public on Dec 21, 2017 |
| Title |
noTF-condition_control-siRNA_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
BJ foreskin fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
viral infection: none
sirna: Non-targeting control #1 (10nM Silencer Select)
|
| Treatment protocol |
Cells were treated with two rounds of transfection with 10nM Silencer Silect siRNAs (Thermo Fisher Scientific) and 2.5 μl Lipofectamine RNAiMAX (Thermo Fisher #13778150), for 3 days per transfection. RNA was harvested at day 6 after the first transfection. For samples in the "hepatic TF" protocol, cells were first infected with lentiviruses delivering the human hepatic transcription factors FOXA3, HNF1A, and HNF4A (MOI 1.25 per virus) the day before the first siRNA transfection (day -1), or 7 days before RNA harvest.
|
| Growth protocol |
Human BJ foreskin fibroblasts were obtained from Stemgent (08-0027) at passage 6 and cultured in Eagle’s Minimum Essential Medium (Sigma M2279) supplemented with 10% fetal bovine serum (FBS, Hyclone SH30071) and 2mM L-glutamine (Gibco) at 37°C and 5% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was purified using the ZR-96 Quick-RNA kit (Zymo Research R1052) and was polyA-selected using Oligo(dT)25 Dynabeads (Thermo Fisher Scientific, #61002).
NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, #E7420S)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
noTF-condition_control-siRNA_pooledReps.bw
|
| Data processing |
Samples were demultiplexed (bcl2fastq using BaseSpace) to produce FASTQ files.
Sequenced reads were aligned to the hg19 Refseq gene model, in a strand-specific manner, using TopHat v2.0.11 (parameters: --b2-very-sensitive --library-type fr-firststrand).
To generate genome coverage tracks, BED files were first pooled between biological replicates. Reads aligning to genomic regions longer than the sequencing length (75 bp), due to spanning of splice junctions, were discarded. Pooled BED files were then converted to BedGraph files using genomeCoverageBed (bedtools v2.20.1) and normalized to the number of millions of reads sequenced, to correct for lane or sample biases. The resulting subtracted BedGraph was converted to a bigWig file using bedGraphToBigWig (v4).
Genome_build: hg19
Supplementary_files_format_and_content: bigWIg
|
| |
|
| Submission date |
Sep 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
|
| Department |
Cell & Developmental Biology
|
| Lab |
Zaret Lab
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE87040 |
Genomic and proteomic resolution of heterochromatin and its restriction of alternate fate genes (RNA-seq) |
| GSE87041 |
Genomic and proteomic resolution of heterochromatin and its restriction of alternate fate genes |
|
| Relations |
| BioSample |
SAMN05785402 |
| SRA |
SRX2173016 |
