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| Status |
Public on Nov 21, 2013 |
| Title |
Parkinson's Disease (PD) patient, post-DBS following 1 hour off electrical stimulation (OFF-Stim), sample 12 |
| Sample type |
SRA |
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| Source name |
Blood leukocytes, PD, post-DBS, off electrical stimulation
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| Organism |
Homo sapiens |
| Characteristics |
cell type: blood leukocytes
disease state: Parkinson's disease
treatment state: post-deep brain stimulation following 1 hour off electrical stimulation
gender: male
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| Treatment protocol |
The captured leukocytes were treated with RNAlater immediately after filtration of the blood and were maintained frozen until RNA extraction.
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| Growth protocol |
Leukocytes were filtered from total blood samples (collected with EDTA vacutainer tubes) with Ambion Inc. LeukoLOCK™ filters within 10 minutes of blood extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using the alternative protocol for extraction of RNA from cells captured on LeukoLOCK™ Filters Using TRI Reagent®. The amount of ethanol was adjusted to include the small RNA fraction. Libraries for deep sequencing were prepared from total leukocyte RNA with the manufacturer's SREK (Small RNA Expression Kit) protocol (Applied Biosystems, Foster City, CA, USA). The DNA libraries were tested using a DNA 1000 Lab Chip on Bioanalyzer 2100. Briefly, the total RNA samples were hybridized with Adaptor Mix A, which is a set of oligonucleotides with a single-stranded degenerate sequence at one end and a defined sequence required for SOLiD sequencing at the other end. The Adaptor Mix A constrains the orientation of the RNA in the ligation reaction such that hybridization with it yields template for SOLiD sequencing from the 5' end of the sense strand. After hybridization, the adaptors are ligated to the small RNA molecules using Ligation Enzyme Mix, which is a mixture of an RNA Ligase and other components. Ligation requires an RNA molecule with a 5'-monophosphate and a 3'-hydroxyl end; therefore, most small RNAs can participate in this reaction, and intact mRNA molecules with a 5' cap structure are excluded. Next, the small RNA population with ligated adaptors was reverse transcribed for each sample, to generate a cDNA library. This was followed by treatment with RNase H to digest the RNA from RNA/cDNA duplexes and to reduce the concentration of unligated adaptors and adaptor by-products. The cDNA libraries were amplified using bar-coded primer sets and 15-18 cycles of PCR. The amplified cDNA library was cleaned up and size selected from gel - PCR products ~105–150 bp are isolated, corresponding to inserts derived from the small RNA population. These contained 90 base pairs of primers and adapter sequences each. The amplified cDNA libraries generated with the SOLiD Small RNA Expression Kit were thereafter ready for attachment to beads at the emulsion PCR step of the SOLiD sample preparation workflow. The slides were analyzed on an Applied Biosystems SOLiD System 3.0 (V3.5). The Applied Biosystems SOLiD sequencer generated 50-base read sequences as .csfasta files and the corresponding quality control (.QUAL) files.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
AB SOLiD System 3.0 |
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| Data processing |
Prior to alignment, all of the reads were subjected to trimming of the 15 end-terminal base pairs using home-made software.
The adapter and primer sequences were further trimmed. These included trimming of the 5' small RNA adapter, discarding of the SOLiD miRNA reverse primer, trimming of the 3' small RNA adapter and removal of the 5' end-terminal nucleotide using CLC bio Genomics Workbench software version 4.0 beta. Of a total of 166,746,207 sequence reads, 71,939,833 were left post-filtering, average length: 21 bp.
The remaining sequence reads were subjected to alignment to miRBase (version 15) human sequences through CLC bio Genomics Workbench software version 4.0 beta.
The reads were further subjected to alignment to the human filter reference sequence database (Ambion, Inc.) of 764 small RNA sequences including snoRNA, tRNA, spliceosomal RNA, etc.
Genome_build: 1) miRBase version 15; 2) human filter reference sequences database (Ambion, Inc.)
Supplementary_files_format_and_content: 1) grouped_miRBase.txt: counts grouped by miRNA name and form (including * forms and sequence); 2) grouped_on_mature_miRBase.txt: counts grouped only by mature miRNA name; 3) unannotated.txt: unannotated sequences identified in the sample; 4) grouped_filter_seq_DB.txt: counts grouped by RNA molecule name (other than miRNA).
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| Submission date |
Oct 05, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Lilach Soreq |
| E-mail(s) |
l.soreq@ucl.ac.uk
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| Phone |
+44 07938689793
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| Organization name |
UCL
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| Department |
Molecular Neuroscience
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| Lab |
Prof. John Hardy
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| Street address |
Queen Square
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| City |
London |
| ZIP/Postal code |
WC1N 3BG |
| Country |
United Kingdom |
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| Platform ID |
GPL9442 |
| Series (1) |
| GSE40915 |
Deep sequencing of the small RNA transcriptome of blood leukocytes from Parkinson's disease patients prior to and following deep brain stimulation (DBS) and as compared with healthy control volunteers |
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| Relations |
| SRA |
SRX192117 |
| BioSample |
SAMN01758864 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1015773_SREK_12_filter_15_end_bp_trim_Small_RNA_sample_grouped_filter_seq_DB.txt.gz |
7.7 Kb |
(ftp)(http) |
TXT |
| GSM1015773_SREK_12_full_15_end_bp_trim_Small_RNA_sample_grouped_miRBase.txt.gz |
7.4 Kb |
(ftp)(http) |
TXT |
| GSM1015773_SREK_12_full_15_end_bp_trim_Small_RNA_sample_grouped_on_mature_miRBase.txt.gz |
5.4 Kb |
(ftp)(http) |
TXT |
| GSM1015773_SREK_12_full_15_end_bp_trim_Small_RNA_sample_unannotated.txt.gz |
3.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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