|
| Status |
Public on Dec 14, 2016 |
| Title |
ChIP dCas9-BFP-DNMT3A (500 ng) + uPA gRNA (500 ng) |
| Sample type |
SRA |
| |
|
| Source name |
Human embryonic kidney
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
chip antibody: anti-HA tag antibody (Cell Signaling, catalog# C29F4, lot# 5 from 12/2014)
|
| Treatment protocol |
Unless stated elsewhere, cells were transfected with gRNAs (total 500 ng) and a CRISPRme expression vector (500 ng) in six-well plates using X-tremeGENE 9 DNA transfection reagent (Roche). For single gRNA or pUC19 control transfections, the amount of plasmid added was equivalent to the total amount of plasmid added for multiple gRNA transfections.
|
| Growth protocol |
Human embryonic kidney HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies), 10% fetal bovine serum (Sigma), 1% penicillin-streptomycin (Sigma), 1X GlutaMAX (Life Technologies).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69506) according to the manufacturer’s instructions. For ChIP, transfected cells were subjected to chromatin immunoprecipitation (ChIP) with a commercially available kit ChIP-IT Express Enzymatic (53009-AF, ActivMotif, distributed by Nordic Biolabs) and an anti-HA tag antibody (C29F4, Cell Signaling) according to the manufacturer's instructions.
Library Preparation was followed by DNA-end repair, 3'-dA overhang and ligation of sequencing adaptors, PCR amplification and size selection (100-300bp, including adaptor sequence).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ChIP DNMT3A
|
| Data processing |
Illumina Casava1.8 software used for basecalling.
ChIP-seq clean reads were mapped to human genome hg19 using SOAP2 with the parameter "-p 4 -v 2 -s 35".
Unique mapping reads was sampled randomly and equally (62723057 reads).
Peaks were called using MACS with P value 1e-3. Sequence motifs enriched within 70 bp of peak summits were identified using MEME-ChIP.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited MACS peak files
|
| |
|
| Submission date |
Dec 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Yonglun Luo |
| E-mail(s) |
alun@biomed.au.dk
|
| Phone |
0045-22411944
|
| Organization name |
Aarhus University
|
| Street address |
Wilhelm Meyers Allé 4
|
| City |
aarhus |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE92261 |
Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA Methylation by dCas9 methyltransferases (CRISPRme) [ChIP-Seq] |
| GSE92311 |
Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA Methylation by dCas9 methyltransferases (CRISPRme) |
|
| Relations |
| BioSample |
SAMN06134707 |
| SRA |
SRX2417121 |
