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| Status |
Public on Dec 03, 2009 |
| Title |
5' NCR from HCV isolated at week 23 from 4x0514 rep2 |
| Sample type |
SRA |
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| Source name |
HCV-from-4x0514_week23
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| Organism |
Hepacivirus hominis |
| Characteristics |
animal id: 4x0514
treatment protocol: locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122
time (weeks of treatment): 23
barcode: MID7
region: region2
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| Extracted molecule |
other |
| Extraction protocol |
The 5’ end of the viral RNA from serum was reverse transcribed into complementary DNA and the cDNA was amplified by hemi-nested polymerase chain reaction (PCR) to obtain a specific PCR product. RNA from 10 μl of serum was used for RT-PCR using RNA UltraSense OneStep Quantitative RT-PCR Kit (Invitrogen) for first round of PCR. The forward primer was 1-20MF (5’-gccagccgcctgatgggggc-3’ with a G/C change at nucleotide 8 to destabilize the step loop structure in the primer. The RT and reverse primer was 274R 5’-tcgcgacccaacactactcggc-3’. The cycles were 60ºC for 30 min, 95ºC for 10 min, and 45 cycles of 95ºC for 15 sec, 60ºC for 1 min, 72ºC for 30 sec with a final cycle of 72ºC for 5 min. One tenth of the PCR product was subjected to a hemi-nested PCR reaction using 1-20MF and 223R (5’-tccaggcattgagcgggtttat-3’) in a Vent Polymerase reaction for 25 cycles at 94ºC for 1 min, 60ºC for 1 min 30 sec, 72ºC for 3 min with a final cycle of 65ºC for 5 min. The PCR product was purified by running on a 1% TAE agarose gel, and by isolating the 223 bp product followed by purification using QIAquick Gel Extraction Kit (Qiagen). The purified PCR products were subjected to deep sequencing by 454 Titanium Sequencing (SeqWright, Houston TX). The samples were first modified to have unique sequence ends with Multiplex Identifiers (MIDs or bar coded).
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| Library strategy |
OTHER |
| Library source |
viral RNA |
| Library selection |
RT-PCR |
| Instrument model |
454 GS FLX |
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| Description |
4x0514-9-17-08
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| Data processing |
A single amplicon was analyzed for variations using the Roche/454 GS Amplicon Variant Analyzer. Barcoded samples were demultiplexed by the software using gsMID tag definitions.
Processed data files include .fasta and .qual files (corresponding to phred quality scores for the fasta files).
Additional processed data available on the Series record include:
AMPLICON.txt, which contains the reference sequence of the amplicon that we have sequenced and the primer sequences
Condensed_all_variants_36x8.txt, which describes frequencies of variations relative to the reference sequence observed in the different samples
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| Submission date |
Nov 06, 2009 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Andreas Petri |
| E-mail(s) |
anp@santaris.com
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| Organization name |
Santaris Pharma
|
| Department |
microRNA
|
| Street address |
Kogle Alle 6
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| City |
Hoersholm |
| ZIP/Postal code |
2970 |
| Country |
Denmark |
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| Platform ID |
GPL9540 |
| Series (1) |
| GSE18919 |
Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection |
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| Relations |
| BioSample |
SAMN02196533 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM468727_region2.MID7.fna.gz |
752.3 Kb |
(ftp)(http) |
FNA |
| GSM468727_region2.MID7.qual.gz |
3.0 Mb |
(ftp)(http) |
QUAL |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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