 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 23, 2017 |
| Title |
239_K562-A |
| Sample type |
SRA |
| |
|
| Source name |
chronic myelogenous leukemia cells
|
| Organism |
Homo sapiens |
| Characteristics |
strain: N/A
|
| Growth protocol |
K562 (a gift from Alexander Stark lab) cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-Glutamine and Pen/Strep (100 U/ml and 100 μg/ml).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
2-4 million cells were fixed for 10 minutes by resuspending the cell pellet in 5 ml full culture medium supplemented with 1% formaldehyde (Sigma). The reaction was quenched by addition of 2 M glycine to a final concentration of 125 mM and incubation for 5 minutes on ice. Cells were pelleted by centrifugation, washed by resuspension in 1 ml of PBS and pelleted again. Then the cells were resuspended in 1.5 ml of lysis buffer and incubated on ice for at least 15 minutes. Cells were pelleted by centrifugation at 2,500g for 5 minutes, resuspended in 100 μl of 1×NEBuffer 3 and pelleted again. The pellet was resuspended in 100 μl of 0.3% SDS in 1×NEBuffer 3 and incubated at 37°C for 1 hour. Then the suspension was diluted with 330 μl of 1× NEBuffer 3 and 53 μl of 20% Triton X-100 (Sigma), and incubated at 37°C for 1 hour to quench SDS. The cells were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 250 μl of 1× DpnII buffer (NEB). 20 μl were taken as a chromatin integrity control, then 600 U DpnII enzyme (NEB) were added, and the chromatin was digested overnight at 37°C with rotation. In the morning 200 more units of DpnII were added and the cells were incubated for additional 2 hours. DpnII was inactivated by incubation at 65°C for 20 minutes. 20 μl was taken as a digestion control. Nuclei were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 100 μl of 1× T4 DNA ligase buffer (Thermo Scientific) and pelleted again. The pellet was resuspended in 500 μl of 1× T4 DNA ligase buffer, and 50 U T4 DNA ligase (Thermo Scientific) were added. The sample was mixed by inversion and incubated with rotation for 4 hours at 16°C and then for 30 minutes at room temperature. Then the nuclei were pelleted by centrifugation at 3,000g for 5 minutes and resuspended in 120 μl of sterile PBS. 20 μl were taken as a ligation control. The rest of the sample was stained with Hoechst, single G1 nuclei were sorted using excitation wavelength 375 nm and forward and side scatter by FACS (FACSAriaIII machine, BD Biosciences) into wells of 12 PCR strips containing 3 μl sample buffer from illustra GenomiPhi v2 DNA amplification kit (GE Healthcare), then they were covered by 5 μl of mineral oil, incubated at 65°C overnight and amplified according to a protocol from Kumar et al, 2008. The amplification was considered positive if the sample contained ≥1 μg DNA, and these samples were submitted for library preparation and sequencing
The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample preparation protocol for Solexa platforms (NEB reagents).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Base calling was performed by VBCF NGS unit
Mapping: iterative mapping (see Imakaev et al, 2012) using bowtie2 with increased stringency parameters: --score-min -L 0.6,0.2--very- sensitive
Read filtering: if two reads map to the same strands, and each side of the read is within 500 bp of any side of the other read, we retained only one copy of the read.
Additional filtering: we separated the genome into 500 bp bins. If any two bins were interacting more than once, only one interaction was counted. Since each region is only present in the single cell up to 4 times (in oocytes), we eliminated all 500 bp bins that form more than 8 unique interactions with other bins.
Filtered reads were then binned at different resolutions (we provide files at 200Kb resolution) using read start as a read position. No normalization was applied to the data.
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: List of all interactions at single contact, 40Kb or 200 Kb resolution
|
| |
|
| Submission date |
Apr 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ilya M. Flyamer |
| E-mail(s) |
flyamer@gmail.com
|
| Organization name |
Friedrich Miescher Institute for Biomedical Research
|
| Lab |
Giorgetti
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE80006 |
Single-cell Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition |
|
| Relations |
| BioSample |
SAMN04622179 |
| SRA |
SRX1685437 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2110094_199_K562_SetA-200kb.csv.gz |
10.8 Kb |
(ftp)(http) |
CSV |
| GSM2110094_199_K562_SetA-40kb.csv.gz |
13.6 Kb |
(ftp)(http) |
CSV |
| GSM2110094_199_K562_SetA-reads.csv.gz |
15.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
