|
| Status |
Public on Dec 16, 2013 |
| Title |
GM15851 |
| Sample type |
SRA |
| |
|
| Source name |
EBV lymphoblastoid cell line
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: PBMC
group: Healthy individual
cell line: GM15851
|
| Biomaterial provider |
http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM15851
|
| Growth protocol |
Epstein Barr virus (EBV)-transformed lymphoblastoid cells were purchased from the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository at the Coriell Institute, Camden, New Jersey, USA. Lymphoblastoid cell lines were grown in RPMI 1640 (PAA) medium with 2 mM L-glutamine and 10% fetal calf serum (PAA) and supplemented with 1x penicillin/streptomycin solution (GIBCO) at 37 °C in 5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The 3C protocol was adapted from Hagege et al. (2007) with some modifications. 1x107 cells was cross-linked with 1% formaldehyde in 10ml culture medium for 10 minutes at room temperature. The reaction was quenched by the addition of glycine to 0.125M. Nuclei were harvested by lysis of the cells in 1ml ice-cold lysis buffer (150mM NaCl, 50mM Tris-HCl (pH 7.5), 5mM EDTA, NP-40 (0.5% v/v), Triton-X-100 (1.0% v/v)) with 1x protease inhibitor cocktail solution (Sigma) plus 0.5mM PMSF. Nuclei were resuspended in 0.5ml EcoRI restriction buffer (NEB) with 0.3% SDS and incubated for 1 hour at 37°C with agitation. Triton X-100 was added to 2% (v/v) and incubated for 1 hour at 37°C with agitation. Cross-linked nuclei were digested overnight at 37°C with 400U EcoRI. Addition of SDS to 1.6% to stop restriction and further incubated at 65°C for 20 minutes with agitation. The reaction was diluted with 7ml ligation buffer with Triton-X-100 and incubated for 1 hour at 37°C prior addition of 100U of T4 DNA ligase (Fermentas) for 4 hours at 16°C and then 30 minutes at RT. Reverse cross-linking was done by adding 600mg Proteinase K and incubated at 65°C overnight. After RNase A digestion, DNA was purified by phenol/ chloroform extraction. The DNA pellet was resuspended in 150 ml 10 mM Tris-Cl (pH7.5).
3C-seq templates were generated as for 3C by using EcoRI. After ligation and purification, the restriction fragments ligated to the anchor region (containing FXN exon 1) were amplified by inverse PCR as described (Simonis et al., 2006). 1 µg of template was amplified per reaction by using Phusion High-Fidelity PCR kit (Finnzymes; Cat.# F-553S) and primers incorporated with different indexes. Primer sequences were listed in Supplementary Table 1. The libraries were processed according to manufacturer’s instructions (Illumina Inc.).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
3C-seq
|
| Data processing |
Illumina CASAVA 1.8.2 software was used for basecalling. The reads were demultiplexed, allowing no mismatches in the index sequence.
Sequencing reads were mapped to hg19 genome using bowtie-0.12.7 with parameters -p 16 -r 100.
Analysis specific to 3C-Seq was performed using r3CSeq Bioconductor package.
Genome_build: hg19
Supplementary_files_format_and_content: bed format files were created by obtaining the normalized values in the form of reads per million per restriction fragment size.
|
| |
|
| Submission date |
Dec 17, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Sanjay Padmakar Khadayate |
| Organization name |
MRC London Institute of Medical Sciences
|
| Street address |
Du Cane Road
|
| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE42961 |
Determination of the spatial organization of the FXN gene locus by using Chromosome Conformation Capture technique coupled with High-throughtput sequencing (3C-seq). |
|
| Relations |
| SRA |
SRX210741 |
| BioSample |
SAMN01831460 |
