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| Status |
Public on Jun 30, 2014 |
| Title |
siRNA1 |
| Sample type |
SRA |
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| Source name |
HUVEC cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HUVEC
mdb capture: Methylated DNA was captured using the MethylCap kit (Diagenode AF-100-0048, Belgium)
tnf treatment: 1 % BSA in PBS for 48 hours
construct: SIRT1 siRNA 1
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| Treatment protocol |
Overexpression of SIRT1 in HuVECs was by transient transfection with the plasmid pCMV6-ENTRY-SIRT1 (Origene) and knockdown of SIRT1 was achieved using two different siRNAs, and compared with a control siRNA, as described previously for Caco-2 cells (Ions et al. 2012). Cells were treated with 0.1 ng/ml tumour necrosis factor alpha (Roche) in 1 % BSA in PBS for 48 hours. Control cells were treated with 1% BSA in PBS.
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| Growth protocol |
HuVECs (passage 3) were seeded into 75-cm2 flasks at a density of approximately 1 x106 cells per flask and maintained at 37_¡C in a humidified atmosphere of 5% CO2 in air in EGMª endothelial growth medium supplemented with & EGMª-2 BulletKitª. All tissue culture reagents were supplied by Lonza. The medium was replaced twice weekly. Experiments were carried out at passage 5-6 in EGMª endothelial growth medium supplemented with 2% (v/v) fetal calf serum (Sigma) and 60 _g/ml gentamycin (Sigma).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from HuVECs using the QIAamp¨ DNA Mini Kit (Qiagen) and quantified by Quant-iTª PicoGreen¨ dsDNA Assay Kit (Invitrogen). Quality of the DNA was assessed using the Nanordrop 1000 (Thermoscientific), OD 260/280 was between 1.8 and 2.0. Enrichment of HuVEC DNA for the methylated fraction was carried out by NXT-DX, The Netherlands. DNA was fragmented to an average length of 200 bp by sonication and analysed using the Agilent 2100 Bioanalyser (Agilent Technologies). Methylated DNA was captured using the MethylCap kit (Diagenode), based on binding to methylated DNA of a recombinant H6-GST-MBD protein then capture on magnetic beads coated with GSH. For each condition (SIRT1 overexpressed by transfection with pCMV6-ENTRY-SIRT1, corresponding vector control, each of the siRNAs that target SIRT1 and control siRNA) six biological replicates were pooled for analysis.
Library preparation is a modification of the ‘multiplexed paired end ChIP protocol’ (Illumina, San Diego, California, USA). The DNA Sample Prep Master Mix Set 1 (NEB E6040) was used in combination with the Multiplexing Sample Preparation Oligo Kit (96 samples, Illumina PE-400-1001). 250ng of fragmented DNA was used for library preparation on Apollo 324 with PrepX-DNA Library Kit (24 samples per kit, 400007) or PrepX-32-DNA Library Kit (96 samples per kit, 400021) according to the kit’s protocol. Library amplification proceeded according to multiplexed paired end ChIP protocol including the indexes from Multiplexing Sample Preparation Oligo Kit. A fragment of 300 bp +/- 50bp was excised from an agarose gel and eluted on a Qiagen Gel Extraction Kit column (Qiagen 28704), then eluted it in 23 μl EB. All samples were adjusted to 10nM and indexed samples were pooled per lane. The paired end (PE) flow cell was prepared according to the Cluster Station User Guide.
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| Library strategy |
MBD-Seq |
| Library source |
genomic |
| Library selection |
MBD2 protein methyl-CpG binding domain |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Alignment - bowtie2 (v2.1.0) options --very-sensitive -X 400
Sort & Index - Picard Tools (v1.99) SortSam.jar (VALIDATION_STRINGENCY=LENIENT MAX_RECORDS_IN_RAM=20000000 CREATE_INDEX=true SORT_ORDER=queryname)
Filter unmapped reads - Picard Tools (v1.99) FilterSamReads.jar (FILTER=includeAligned)
Identify differentially methylated regions - MEDIPS (Bioconductor)
Find differentially methylated regions in/near GENCODE (release 16) genes - BedTools (v 2.17.0) - intersectBed
Genome_build: hg19
Supplementary_files_format_and_content: text table, 100bp windows with bam counts, RPKM, genome-wide
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| Submission date |
Jan 14, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Simon Cockell |
| E-mail(s) |
simon.cockell@newcastle.ac.uk
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| Organization name |
Newcastle University
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| Street address |
Framlington Place
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| City |
Newcastle |
| ZIP/Postal code |
NE2 4HH |
| Country |
United Kingdom |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE54072 |
SIRT1 affects DNA methylation of polycomb group protein target genes (PGCTs), a hotspot of the epigenetic shift observed in ageing. |
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| Relations |
| BioSample |
SAMN02584331 |
| SRA |
SRX426496 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1306805_siRNA1_processed.txt.gz |
290.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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