|
| Status |
Public on Jul 16, 2015 |
| Title |
TET1-A_-DOX |
| Sample type |
SRA |
| |
|
| Source name |
Flp-In™ T-REx™ 293
|
| Organism |
Homo sapiens |
| Characteristics |
transgene: TET1
treatment: uninduced
cell line: HEK293
|
| Treatment protocol |
Doxycycline was used to induce the expression of the integrated TET1 copy.
|
| Growth protocol |
Cells were cultured in DMEM supplemented with 10% FCS, 1% Pen/Strep and appropriate concentrations of selective antibiotics.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using the AllPrep DNA/RNA/Protein Mini kit (Qiagen)
Genomic DNA was fragmented overnight at 37°C with a hydroxymethyl-insensitive enzyme, MspI, and purified using the DNA Clean and Concentrator kit (Zymo Research). Modified Illumina TruSeq P5 and P7 adapters containing 5’-CG overhangs were ligated onto the digested DNA using T4 DNA ligase (2 hours at 16°C). Libraries were then strand extended at 72°C with Taq DNA Polymerase. The adapters were designed to regenerate the 5’-CCGG site at the P5 junction while the P7 adapter generates a 5’-TCGG junction, making it insensitive to MspI digestion. Adapterized libraries were treated with ß-glucosyltransferase to label 5-hmC modifications and purified using the DNA Clean and Concentrator kit. The glucosylated libraries were then subjected to an overnight MspI digestion at 37°C, cutting any fragments not containing a glucosyl-5hmC site at the P5 CCGG junction. After incubation, the libraries were size-selected from 100bp to 500bp and purified using the ZymoClean Gel DNA Recovery Kit (Zymo Research). The fragments were amplified using OneTaq 2X Master Mix (NEB), and the PCR conditions include an initial denaturation of 94°C for 30 sec followed by 12 cycles of 94°C for 30 sec, 58°C for 30 sec, and 68°C for 1 min. Fragments containing 5-hmC were positively selected during PCR amplification with adapter-specific indexing primers whereas fragments lacking glucosylated-5-hmC at the P5 junction were cleaved and, therefore, not amplified by PCR. Amplified libraries were purified using the DNA Clean and Concentrator kit, and multiplexed using equal volume of the libraries. All adapters and primers used were synthesized by Integrated DNA Technologies.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
5hmC data from TET1-A clone (TET1#1), not induced
|
| Data processing |
Library strategy: RRHP library
Sequence reads from RRHP libraries were first processed to trim off the low quality bases and the P7CG adapter at the 3’ end of the reads. Reads were then aligned to the hg19 reference genome using the Bowtie default parameters and --best. Aligned reads with the MspI tag (CCGG) were counted.
Genome_build: hg19
Supplementary_files_format_and_content: bigBed files with 5hmC tracks
|
| |
|
| Submission date |
Apr 07, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Bernhard Horsthemke |
| E-mail(s) |
bernhard.horsthemke@uni-due.de
|
| Organization name |
Institut fuer Humangenetik
|
| Department |
Universitaetsklinikum Essen
|
| Street address |
Hufelandstrasse 55
|
| City |
Essen |
| ZIP/Postal code |
D-45122 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE67621 |
Effect of TET1 overexpression on hydroxymethylcytosine levels of HEK293 cells |
|
| Relations |
| BioSample |
SAMN03463638 |
| SRA |
SRX980982 |
