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| Status |
Public on May 25, 2013 |
| Title |
Ago2 PAR-CLIP |
| Sample type |
SRA |
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| Source name |
LCL cell line established using EBV B95-8 BACmid lacking ebv-mir-BHRF1-2 (D2)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LCL-BACD2
antibody: Anti-Ago2 (clone 9E8, Millipore cat#04-642)
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| Growth protocol |
The established lymphoblastoid cell line (LCL-BAC-D2) is infected with an EBV B95-8 BACmid (BAC) and was analyzed four months post-infection. LCL-BAC-D2 was maintained in RPMI 1640 medium supplemented with 15% FBS and 10 μg/mL gentamicin (GIBCO). LCL-BAC-D2 (mutationally inactivated for miR-BHRF1-2 expression) is infected with an EBV miRNA-mutant virus described in (Feederle, et. al., J. Vir., 2011), and were generated from the same donor as LCL-BAC, LCL-BAC-D1, and LCL-BAC-D3 (Skalsky, et. al., PloS Path., 2012; GEO GSE41437).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For UV crosslinking, ~4 L of cells were cultured in R-15 containing 100 microMolar 4-thiouridine overnight and collected by centrifugation. Cells were washed once with ice-cold PBS and irradiated on ice at UV 365 nm in a Stratalinker 2400 (Stratagene). Cross-linked cells were collected by centrifugation at 500×g for 5 min.
The pellets of cells crosslinked with UV 365 nm were resuspended in NP40 lysis buffer and incubated on ice for 10 min. The cell lysate was cleared by centrifugation at 13,000g. RNase T1 (Fermentas) was added to the cleared cell lysates and the reaction mixture was incubated at 22ºC for 15 min and subsequently cooled for 5 min on ice before addition of antibody-conjugated magnetic beads. The recovered RNA was carried through a cDNA library preparation protocol originally described for cloning of small regulatory RNAs (Hafner et al., 2008). The first step, 3' adapter ligation, was carried out as described on a 20 ul scale using 10.5 ul of the recovered RNA. UV 365 nm crosslinked sample RNAs were ligated to Illumina sequencing adapter sets and reversed transcribed to cDNA. Following PCR amplification of the cDNA, products were gel-extracted and sequenced on the Illumina GAIIX sequence analyzer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Ago2 ribonucleoprotein complexes
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| Data processing |
Library strategy: PAR-CLIP
PAR-CLIP: Libraries were stripped of the 3’ adapter sequence and 5' barcode using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Reads that were less than 13 nt in length or contained an ambiguous nucleotide were discarded. The small RNA files are in a tab-delimited form: SEQUENCE READCOUNT. The Ago2 PARCLIP reads were aligned to the human genome (hg19), with up to 2 mismatches allowed, by the Bowtie algorithm (Langmead et al., 2009). Mapped locations were only reported for those with the minimum number of observed mismatches for each read. All T to C mismatches between the RNA fragment and the genome were subtracted from the mismatch count for each mapped location. After subtraction only reads that mapped to a single genomic location at the minimum number of mismatches were used for further analysis. The location that a read mapped, relative to a known transcript, was determined based on the ENSEMBL v57 database 1 (Hubbard et al., 2007). If a read mapped to a location representing multiple categories, it was reported to belong to the category based on the following order of preference: 3’UTR, coding sequence, 5’UTR, intron, non-coding RNA, intergenic. Defining microRNA interaction sites by PARalyzer: Overlapping reads were grouped together for further analysis. A group must have contained ≥5 reads with conversions at two or more locations. Separate kernel density estimates were calculated across all positions based on read counts both with and without T to C conversions, as long as there was a minimum read depth of 5 at a position to ensure a robust density estimate. Clusters (*.bed files) were defined as regions for which the conversion density was greater than the non-conversion density for at least 5 consecutive nucleotides, and extended until the group read depth fell below 5. Following filtering (see supplemental methods), clusters were scored by a cross-linking index (CLI) = log2(1+T to C conversion events for all reads within a cluster). PARalyzer is available at http://www.genome.duke.edu/labs/ohler/research/PARalyzer
RNA-seq: RNA-seq data was aligned and quantified using RSEM with reference genome and transcript annotation [B. Li and C. Dewey (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323]. All necessary transcript annotation, genomic sequence, and alignment indices were downloaded from the Illumina iGenomes collection for Ensembl build GRCh37. Differential analysis was performed using EBseq within RSEM [Leng, N., J.A. Dawson, J.A. Thomson, V. Ruotti, A.I. Rissman, B.M.G. Smits, J.D. Haag, M.N. Gould, R.M. Stewart, and C. Kendziorski. EBSeq: An empirical Bayes hierarchical model for inference in RNA-seq experiments, UW-Madison Department of Biostatistics and Medical Informatics Paper, Bioinformatics 2012].
Genome_build: hg19
Supplementary_files_format_and_content: bed and expression
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| Submission date |
May 03, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Neelanjan Mukherjee |
| E-mail(s) |
neelanjanmukherjee@gmail.com
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| Phone |
3094061817
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| Organization name |
MDC Berlin
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| Department |
BIMSM
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| Lab |
Uwe Ohler
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| Street address |
Robert-Rössle-Straße 10
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| City |
Berlin |
| State/province |
---- |
| ZIP/Postal code |
13125 |
| Country |
Germany |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE46611 |
MicroRNA Target Site Identification by Integrating Sequence and Binding Information |
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| Relations |
| BioSample |
SAMN02116962 |
| SRA |
SRX273682 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1133252_LCLBACD2_PC.bed.gz |
20.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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