|
| Status |
Public on Aug 17, 2017 |
| Title |
BRAF OIS 2 - Dam Only |
| Sample type |
SRA |
| |
|
| Source name |
Tig3ET cell line (diploid embryonic lung fibroblasts expressing the ecotropic receptor to allow infection with ecotropic retroviruses and hTERT to prevent replicative senescence)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Tig3
day treament: 10
cell stage: oncogene induced senescence
transducer: Dam lentiviruses
|
| Treatment protocol |
Human diploid fibroblast Tig3 cells expressing the ecotropic receptor, hTERT, and the (pBabe-hygro) empty vector, were transduced with empty vector (cycling). The infected population was selected using puromycin 1μg/ml. Eight days after retroviral infection (a time point at which cells have entered senescence) or after serum starvation, cells were transduced with Dam encoding lentiviruses. Twenty fours hours after transduction, cells were refreshed with medium supplemented with 0.5 μM Sh. After 24 hour of induction cells were harvested and processed for DamID sequencing.
Cells were maintained for 9 days after the treatment specified in Treatment
|
| Growth protocol |
Cells were propagated in DMEM (GIBCO) supplemented with 9% fetal bovine serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using the using Gentra Puregene Cell Kit according to the manufacturer’s instructions. 0.5 mg of gDNA was subjected to digestion with DpnI restriction enzyme, followed by ligation to a double-stranded adaptor. The adaptor-ligated fragments were subsequently PCR-amplified using primers identical to the adaptor sequence and the 5’ TC nucleotides of the gDNA fragment. Then the methylPCR products were cleaned up using the QIAquick PCR product purification kit. Then DNA fragments were end-repaired using the END ITTM end repair kit (Epicentre) according to manufacturer’s instruction. The end-repaired DNA was A-tailed by incubating with 0.4 mM ATP in the presence of 25U of klenow fragment (NEB) at 37°C for 30 min, then, 75°C for 20 min.
DNA was subsequently purified using AMPureXP beads (Agencourt Biosciences) and ligated to the Illumina Y adaptor in a reaction containing 2.5 mM Illumina Y adaptor and 2.5 U of T4DNA ligase and incubated at 16°C for 16 hours, followed by inactivation at 65°C for 10 min. The adaptor ligated DNA was purified with AMPureXP beads. 100 ng of purified DNA was amplified by 12 cycles in a reaction containing Illumina constant primer and Illumina index primer at a final concentration of 2.5 mM, and 10 ml of 2x MyTaq mix in a final volume of 20 ml. Samples were then pooled, bead purified and size-selected.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Sample name: 3211_11_166_D_1_10_40_CATCAA_L007_R1_001
|
| Data processing |
Reads were demultiplexed using bcl2fastq 1.8.4 with 1 mismatch allowed
DamID adaptors were trimmed from reads with cutadapt
Sequencing reads were mapped against the human genome build hg19 using Bowtie2 with parameters --local --phred33
Reads were filtered for those that mapped with qval 15 and unique mapping
Reads which aligned to GATC fragments were summarised as counts
Then summarised into 50kb bins as observed reads over expected in each bin
Genome_build: hg19
Supplementary_files_format_and_content: The file readcounts.txt contains the absolute gene expressions values for each of the samples.
Supplementary_files_format_and_content: OIS_LAD_CdG_GenomeResearch_Annot.csv: Lmnb1-Dam and DamOnly reads were binned into 50k regions and then rations of reads calculated to determine Lamin Associated Domains. Scripts for this are found at https://github.com/cadegraaf/DamIdSeq
|
| |
|
| Submission date |
Jan 06, 2016 |
| Last update date |
Jan 06, 2020 |
| Contact name |
Carolyn A de Graaf |
| E-mail(s) |
degraaf@wehi.edu.au
|
| Organization name |
Walter and Eliza Hall Institute
|
| Street address |
1G Royal Parade
|
| City |
Parkville |
| ZIP/Postal code |
3052 |
| Country |
Australia |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE76594 |
Massive reshaping of genome - nuclear lamina interactions during oncogene induced senescence (DamID-seq) |
| GSE76605 |
Massive reshaping of genome - nuclear lamina interactions during oncogene induced senescence |
|
| Relations |
| BioSample |
SAMN04386868 |
| SRA |
SRX1520587 |
