|
| Status |
Public on Jun 19, 2020 |
| Title |
TET2_4R_TET2i_Bisulfite_Padlock_seq |
| Sample type |
SRA |
| |
|
| Source name |
TET2_TET2i_Bisulfite_Padlock_seq
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-SY5Y
sirna: TET2
transfection hours: 72
replicate: Technical Replicate
|
| Treatment protocol |
SH-SY5Y cell were transfected with 100 nM of either human TET2 siGENOME SMARTpool siRNA or a non-targeting siGENOME Control#2 siRNA (Dharmacon), using X-tremeGENE siRNA Transfection Reagent (Sigma-Aldrich) according the manufacturer’s instructions
|
| Growth protocol |
SH-SY5Y cells were cultured in 10 % FBS (Gibco) RPMI (Thermo Fisher Scientific)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After 72 h post-transfection, genomic DNA was extracted from the SH-SY5Y cells by standard phenol-chloroform extraction methods.
Libraries were prepared using a targeted bisulfite sequencing approach known as bisulfite padlock probes sequencing. Padlock probes were designed to human brain enhancers and promoters (NIH Roadmap ChromHmm) and synthesized using a programmable microfluidic platform (Custom Array, Inc.). Genomic DNA was bisulfite converted and purified using the EZ DNA Methylation-Lightning Kit (Zymo Research). The bisulfite-converted DNA (200 ng) was then hybridized to the padlock probes (1.5 ng). PfuTurbo Cx (Agilent Technologies) was used to extend across the target regions and circularization was completed using Ampligase (Epicentre). Non-circularized DNA was digested using an exonuclease cocktail and the remaining circularized DNA was amplified using a common linker sequence in the padlock probe. Libraries were PCR amplified, pooled in equimolar amounts, purified by QIAquick Gel Extraction kit (Qiagen) and quantified by qPCR (Kapa Biosystems) on a ViiA 7 Real-time PCR system (Applied Biosystems). Next-generation sequencing of the libraries was performed on an Illumina HiSeq 2500 machine on HiOutput mode.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
72 hrs Dharmacon TET2 siGENOME SMARTpool
|
| Data processing |
Libraries sequenced on the HiSeq 2500 platform (Illumina) using custom sequencing primers.
Adapter sequences were removed from fastq files using TrimGalore (v0.4.4).
The remaining sequencing reads were then aligned to the target reference genome (GRCh37/hg19) indexed by Bismark (v0.17.0).
DNA methylation calls were counted using Bismark (v0.17.0).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Each txt file contains the following information in order: chromosome #, genomic coordinate, strand, unmethylation count, methylation count, CHH/CHG/CG context of cytosine, exact sequence of the previous context
|
| |
|
| Submission date |
Aug 02, 2019 |
| Last update date |
Jun 20, 2020 |
| Contact name |
Lee Marshall |
| E-mail(s) |
lee.marshall@vai.org
|
| Phone |
616 920 9824
|
| Organization name |
Van Andel Research Institute
|
| Department |
Center for Neurodegenerative Science
|
| Lab |
Viviane Labrie
|
| Street address |
333 Bostwick Ave NE
|
| City |
Grand Rapids |
| State/province |
MI |
| ZIP/Postal code |
49503 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE135333 |
Epigenomic analysis of Parkinson’s disease neurons identifies TET2 loss as neuroprotective [TET2i_Bisulfite_Padlock_seq] |
| GSE136010 |
Epigenomic analysis of Parkinson's disease neurons identifies TET2 loss as neuroprotective |
|
| Relations |
| BioSample |
SAMN12477076 |
