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| Status |
Public on Jul 31, 2014 |
| Title |
C8166 NL4-3 Day 3 RISC PAR-CLIP |
| Sample type |
SRA |
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| Source name |
C8166 Cell Line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: C8166
hiv-1 strain: NL4-3
cell type: CD4+ T-cell Line
length of infection (days): 3
rip antibody: Abcam AB57113
illumina truseq index number: 6,7
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| Treatment protocol |
Forty-eight hours postinfection, cells were labeled with 100 µM 4-thiouridine (Sigma) for 16 h. Cells were washed with PBS and cross-linked using a Stratagene UV Stratalinker 2400 at 365 nm. The samples were then processed as previously described (Gottwein et al, 2011, Cell Host Microbe 10:515-526). RISC cross-linked RNAs were isolated using anti-Ago2 antibody (Abcam AB57113). (This antibody also recognizes Ago1 and Ago3 [data not shown].) Cloning of isolated small RNAs was performed using the TruSeq small RNA cloning kit (Illumina).
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| Growth protocol |
Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma) supplemented with 10% FBS
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| Extracted molecule |
total RNA |
| Extraction protocol |
TRI-zol, PAGE fractionation ~15-40nt
RNA libraries were prepared according to standard Illumina Truseq protocols
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RISC-associated RNAs isolated from RISC PAR-CLIP. Two separate PCR amplifications were sequenced on separate lanes. Sequencing data from both lanes were combined for analysis after initial read trimming and processing.
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| Data processing |
Casava1.8.2 software used for basecalling.
Reads <15nt were discarded, adaptors trimmed, and low quality reads discarded using the FastX tool kit and the following pipeline: fastq_quality_filter -Q33| fastq_to_fasta -Q33 | fastx_clipper -a TruSeq-Index# -l 15 –c | fastx_collapser
Bowtie v.0.12.7, with the following options: –a --best --strata –m 25, was used to remove adapters and size-fractionation markers with no variant nucleotides for all libraries but the PAR-CLIP libraries. The aforementioned parameters were kept throughout the pipline
Total small RNA and RISC RIP-Seq reads were sequentially filtered by aligning to the viral genome (1mismatch, negative sense alignments allowed), then human pre-miRNAs from miRbasev19 (1mismatch), EnsemblncRNAv70 Homo sapiens (1mismatch), fRNAdbv3.4 Homo sapiens (1mismatch), then human genome 19 (2mismatches, negative sense alignments allowed)
PAR-CLIP reads were aligned to a reference of the human genome combined with either the near-full length viral genome or the 3'LTR of each virus to avoid ambiguous alignments occuring with both annotated LTRs on the same sequence.
Genome_build: hg19
Supplementary_files_format_and_content: Processed data are in a tab-delineated BED format presented as chromosome/gene, starting location for aligned read relative to the start of the gene beginning at 1, ending position of the aligned read, aligned reads per million of total library reads, and the strand (+ or -) that the read aligns to. Processed data files include alignemnts to HIV, miRbase human pre-miRNAs v19, Ensembl human ncRNAs v70, fRNAdb human ncRNAs v3.4, human genome 19 alignments, human+viral genome PARCLIP alignments and human+viral genome Nef+U3 PARCLIP alignments.
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| Submission date |
Jul 30, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Adam Wesley Whisnant |
| E-mail(s) |
adamwhisnant88@gmail.com
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| Phone |
9196845656
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| Organization name |
Duke University
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| Department |
Molecular Genetics and Microbiology
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| Lab |
Bryan R. Cullen
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| Street address |
213 Research Drive 427 CARL Bldg
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE59944 |
In depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms |
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| Relations |
| BioSample |
SAMN02947363 |
| SRA |
SRX666580 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1462572_HCCcolNL43PAR.bed.gz |
11.9 Mb |
(ftp)(http) |
BED |
| GSM1462572_HCCcolNL43U3R.bed.gz |
11.9 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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