|
| Status |
Public on Aug 26, 2016 |
| Title |
4Cm TALE silenced cells rep3 |
| Sample type |
SRA |
| |
|
| Source name |
K562 with B2M:tdTomato transgene
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
|
| Treatment protocol |
K562 cells were treated with a cocktail of three plasmid each encoding for a different ATR. We used 1 ug of palsmid encoding for D3A-based ATR and then normalized the amount of the others plasmids to obtain a 1:1 molar ratio. For dCas9 based ATR treatment we used 250 ng of plasmid encoding for sgRNA.
|
| Growth protocol |
K562 cells were grown in IMDM (Sigma) 10% Fetal Bovine Serum (EuroClone) and 1% Pennicillin/Streptomycin (100 U/ml final concentration; EuroClone). Cells were routinely splitted every 3 days at a concentration of 10^5 cells/ml without allowing them to reach concentration higher than 8^105 cells/ml
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted with Qiagen Kit (Cat.13343) according to manifacturer's instructions.
500 ng of genomic DNA was sonicated with E220 COVARIS ultrasonicator (Covaris) and sequencing libraries were prepared using NextFlex Methylseq kit 1 (Bioo Scientific). After adapter ligation step, samples were pooled and immunoprecipitated using a monoclonal antibody directed against 5-methylcytosine (MagMeDIP kit, Diagenode). Enriched and control libraries (not immumoprecipitated) were purified using the IPure kit (Diagenoide). Enrichment efficiency was evaluated by qPCR using provided internal controls (spiked-in DNA from A. thaliana). Libraries were amplified following NextFlex Methylseq kit 1 protocol
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
4Cm
|
| Data processing |
Reads were aligned to reference genome using bwa mem v0.7.5
Duplicates were processed with Picard MarkDuplicates
Peaks were identified using MACS2 (callpeak -g hs --keep-dup=1 --llocal 500000 --slocal 0 --broad)
Peaks from multiple samples were merged using bedtools multiinter -cluster; counts over merged peaks were obtained using bedtools multibamcov
Genome_build: hg19
Supplementary_files_format_and_content: broadPeak. Features identified for MeDIP-seq samples
Supplementary_files_format_and_content: abundance measurements: raw read counts over merged features (meDIP-seq) or genes (RNA-seq)
|
| |
|
| Submission date |
May 24, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Davide Cittaro |
| E-mail(s) |
[email protected]
|
| Organization name |
Ospedale San Raffaele
|
| Department |
Center for Traslational Genomics and Bioinformatics
|
| Street address |
via Olgettina 58
|
| City |
Milano |
| ZIP/Postal code |
20138 |
| Country |
Italy |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE81826 |
Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing |
|
| Relations |
| BioSample |
SAMN05171595 |
| SRA |
SRX1797745 |
