 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 02, 2016 |
| Title |
tSBE(6+2-Vp64) norm biological replicate 1 technical replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
5C tSBE(6+2-Vp64)
|
| Organism |
Mus musculus |
| Characteristics |
tissue: transfected 46c ESCs
cell type: Stem cells
|
| Treatment protocol |
Cells were fixed with 1% formaldehyde for 10 min at room temperature. Crosslinking was stopped with glycine (125 mM final concentration), and incubated for 5 min at room temperature followed by 15 min on ice. Cells were centrifuged at 400 g for 10 min at 4°C. Supernatants were removed and cell pellets were flash frozen on dry ice.
|
| Growth protocol |
GMEM+ Supplement for ESCs, N2B27 for 7 days for NPCs
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
10-20 million fixed cells were incubated for 15 min on ice in 200 ml of lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP40, supplemented with fresh protease inhibitor cocktail). Cells were then disrupted on ice with a dounce homogenizer (pestle B; 2 x 20 strokes), cell suspensions transferred to eppendorf tubes and centrifuged 5 min at 2000 g. Supernatants were removed, the cell pellets washed twice with 100 ml of 1X CutSmart buffer (NEB) and the cell pellet resuspended in 100 ml of 1X CutSmart buffer, and divided into two eppendorf tubes. 1X CutSmart buffer (337 ml) was added to each tube, and the mixture was incubated 10 min at 65°C with 0.1% SDS. 44 ml of 10% Triton X-100 were added before overnight digestion with 400 Units HindIII. The restriction enzyme was then inactivated by adding 86 ml of 10% SDS, and incubation for 30 min at 65°C. Samples were then individually diluted into 7.62 ml of ligation mix (750 ml 10% Triton X-100, 750 ml 10X ligation buffer, 80 ml 10 mg/ml of BSA, 80 ml 100 mM ATP and 3000 Cohesive end Units of T4 DNA ligase) and incubated at 16°C for 2 hours. 3C libraries were incubated overnight at 65°C with 50 ml Proteinase K (10 mg/ml), and with an additional 50 ml Proteinase K the following day for 2 hours. The DNA was purified by one phenol and one phenol-chloroform extraction, and precipitated with 0.1 volume (800 ml) of 3 M NaOAc pH 5.2 and 2.5 volumes of cold EtOH (20 ml). After at least 1 h at -80°C, the DNA was centrifuged for 25 min at 20,000 g at 4°C, and the pellets washed with cold 70% EtOH. DNA was resuspended in 400 ml of TE pH 8.0, and transferred to eppendorf tubes for another phenol-chloroform extraction and precipitation with 40 ml of 3 M NaOAc pH 5.2 and 1.1 ml of cold EtOH. DNA was recovered by centrifugation, and washed eight times with cold 70% EtOH. Pellets were then dissolved in 100 ml TE pH 8.0, and incubated with RNAse A (1 ml at 10 mg/ml) for 15 min at 37°C.
3C libraries were denatured and annealed to 5C primers. 5C oligonucleotide were designed upstream (FORWARD) or downstream (REVERSE) HindIII restriction sites. Annealing was followed by ligation with Taq DNA ligase. Ligated 5C primers were then amplified with the A-key and P1-key primers to generate the 5C libraries. 5C libraries were then prepared for sequencing onto Ion 316™ Chips with an Ion PGM™ Sequencer following either the Ion Xpress™ Template Kit v2.0, and Ion Sequencing Kit v2.0 protocols, or the Ion PGM™ Template OT2 200 Kit, and Ion PGM™ Sequencing 200 Kit v2.0 protocols as recommended by the manufacturer’s instructions (Life TechnologiesTM). Libraries were prepared according to Ion Torrent's instructions
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Library strategy: 4C-Seq
The sequencing data was processed through a Torrent 5C data transformation pipeline on Galaxy (https://main.g2.bx.psu.edu/). The data was mapped against a customized reference file with TMAP. The reference file contained a list of all possible contacts between Forward and Reverse 5C primers covering our regions.
The data was then filtered to remove low-quality reads (MAQ quality score of lower than 30), reads aligning more than two nucleotides away from the reference sequence start site, and reads which do not contain HindIII restriction sites.
5C data was normalized by dividing the number of reads of each 5C contact by the total number of reads from the corresponding sequence run multiplied by 10^3.
Genome_build: mm9
Supplementary_files_format_and_content: This analysis generates a matrix file containing the interaction frequencies
|
| |
|
| Submission date |
Nov 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Nezha Suzanne Benabdallah |
| E-mail(s) |
nsbenabdallah@gmail.com
|
| Organization name |
German Cancer Research Center, DKFZ
|
| Department |
Functional and Structural Genomics
|
| Lab |
Soft-Tissue Sarcoma
|
| Street address |
Im Neuenheimer Feld 581 (TP4)
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18635 |
| Series (2) |
| GSE89388 |
A new model for long-range chromatin reorganization linked to enhancer activation [5C] |
| GSE89557 |
A new model for long-range chromatin reorganization linked to enhancer activation |
|
| Relations |
| BioSample |
SAMN05960856 |
| SRA |
SRX2314347 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2367832_tSBE_6+2_-norm-rep1.txt.gz |
220.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
