|
| Status |
Public on Mar 31, 2015 |
| Title |
RPMI_Goat_IgG |
| Sample type |
SRA |
| |
|
| Source name |
Human embryonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SA121
cell type: Human embryonic stem cells
treatement: mock
chip antibody: goat IgG (DAKO)
|
| Treatment protocol |
Confluent cultures were treated with with 3mM CHIR99021 (CHIR), or 3mM CHIR99021+ 10mM SB431542 (CHIR+SB) in RPMI-1640 for 6h.
|
| Growth protocol |
hESC were culture in DEF-CS media (Cellartis).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed for 15 min in culture media containing 1% formaldehyde and were processed for ChIP as in Dietrich et al. (2007) EMBO J 26: 1637–1648.
DNA from three parallel ChIPs were pooled and 10 ng was used for making ChIP-seq libraries. Libraries were generated according to Illumina recommendations and sequencing was done on a Genome Analyzer II (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
High quality reads (Chastity score .= 0.6) were aligned to the mouse genome (mm9) using Bowtie allowing up to two mismatches within the first 32 bases. Reads not aligning uniquely to the mouse genome were removed.
Aligned datasets were filtered for multiple reads at the same position occurring after PCR amplication. Where more than one read mapped to the same position and at the same strand duplicates were removed.
The reads were extended to 250 bp length, and the number of reads at a given position was counted and saved as a fixedStep wig file with 100 bp resolution. Values are denominated in fragments pr. Mreads pr. kbp.
Prior to peak finding the most abundant DNA-fragment size for the sample and IgG control was determined by finding the distance (that as a minimum was two times the read length) between the two strands with most correlation according to the Phantom Peak Coeefficient method (by Anshul Kundaje, akundaje@stanford.edu).
Peaks were identified using ChIP-seq from unspecific IgG antibodies as a negative control. IgG antibodies were raised in the same species as the antibodies used for the samples. Each dataset was divided into 100 bp windows, and the reads within each window counted genome-wide. The distribution of reads within the windows was fitted to the Poisson distribution with the least root-mean-square deviation. Thresholds were set automatically for both the sample and negative control at the level where the number of expected reads were 0,001 and 0,1 times the observed number of reads or less, respectively. Based on this, the threshold for the sample was set to 20 reads and the threshold for the negative sample was set to 11 reads. The genome was analysed in 100 bp windows, and windows that both had a number of reads in the sample at or above the sample threshold and had fewer reads than the negative threshold were scored positive and their positions saved. This analysis was repeated 4 times - each time the windows were shifted 25 bp. Windows within 100 bp of each other and overlapping windows were merged.- For each region in the resulting the borders were refined by sliding a window of 100 bp from one windowsize upstream to downstream of the temporary border. The exact position where the number of sample reads within the window fell below the sample threshold, or the number of negative reads within the window came above the negative threshold, was defined as new new border of that region.
Genome_build: hg19
Supplementary_files_format_and_content: Txt: tab separated list of peaks from peak-calling, wig: fixedStep Wiggle file containing densities with 100 bp resolution of aligned and filtered reads.
|
| |
|
| Submission date |
Jun 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Karen Schachter |
| E-mail(s) |
karen.schachter@sund.ku.dk
|
| Organization name |
University of Copenhagen
|
| Department |
The Danish Stem Cell Center (DanStem)
|
| Lab |
Semb lab
|
| Street address |
Blegdamsvej 3B, building 6.4.24
|
| City |
Copenhagen N |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE58476 |
β-catenin regulates primitive streak induction through collaborative interactions with SMAD2/3 and OCT4 |
|
| Relations |
| BioSample |
SAMN02854556 |
| SRA |
SRX596339 |
