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Sample GSM4006434 Query DataSets for GSM4006434
Status Public on Jun 19, 2020
Title TET2_5_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: Biological Sample
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
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
BioSample SAMN12477075

Supplementary file Size Download File type/resource
GSM4006434_TET2_5_Cxreport.txt.gz 40.7 Mb (ftp)(http) TXT
Raw data are available in SRA
Processed data provided as supplementary file

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