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Sample GSM1462574 Query DataSets for GSM1462574
Status Public on Jul 31, 2014
Title TZM-bl ami BaL Day 3 RISC PAR-CLIP
Sample type SRA
 
Source name TZM-bl Cell Line
Organism Homo sapiens
Characteristics cell line: TZM-bl
hiv-1 strain: WT/BaL
cell type: Cervical Epithelial Cell Line, HeLa derivative transduced with shRNAs against HIV-1
length of infection (days): 3
rip antibody: Abcam AB57113
illumina truseq index number: 5
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).
Growth protocol Dulbecco’s modified Eagle medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS)
Extracted molecule total RNA
Extraction protocol TRI-zol, PAGE fractionation ~15-40nt
RNA libraries were prepared according to standard Illumina Truseq protocols
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Description RISC-associated RNAs isolated from RISC PAR-CLIP
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.
 
Submission date Jul 30, 2014
Last update date May 15, 2019
Contact name Adam Wesley Whisnant
E-mail(s) adamwhisnant88@gmail.com
Phone 9196845656
Organization name Duke University
Department Molecular Genetics and Microbiology
Lab Bryan R. Cullen
Street address 213 Research Drive 427 CARL Bldg
City Durham
State/province NC
ZIP/Postal code 27710
Country USA
 
Platform ID GPL11154
Series (1)
GSE59944 In depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms
Relations
BioSample SAMN02947365
SRA SRX666582

Supplementary file Size Download File type/resource
GSM1462574_TZMblamiPCBaLPAR.bed.gz 41.0 Mb (ftp)(http) BED
GSM1462574_TZMblamiPCBaLU3R.bed.gz 41.0 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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