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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 02, 2017 |
| Title |
iCLIP-seq of Nacc1 in N2A cells, replicate 2 |
| Sample type |
SRA |
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| Source name |
Neuroblastoma cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: N2A
antibody: Nacc1 antibody (abcam ab29047)
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| Treatment protocol |
Crosslinking (0.4 J/cm2) was done at 254 nm with a Stratalinker 1800.
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| Growth protocol |
Mouse neuroblastoma (N2A) cells were grown in DMEM supplemented with 10% FBS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin, and maintained at 37°C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Lysates generated from the crosslinked cells were treated with Turbo DNase (Ambion) and RNase I (1:500; Ambion) for 5 min at 37°C to digest the genomic DNA and trim the RNA to short fragments of an optimal size range. A total of 2% input material was saved to prepare size-matched control libraries. RNA-protein complexes were immunoprecipitated using 100 μl of protein G Dynabeads (Life Technologies) and 10 μg of anti-Nac1 (Abcam). Following stringent high salt washes, the immunoprecipitated RNA was 5' end-labeled using radioactive 32P isotopes. Pre-adenylated adaptors were ligated to the 3' end using the conditions reported by van Nostrand et al. (2016; PMID: 27018577). The immunoprecipitated complexes were separated with SDS-PAGE and transferred to a nitrocellulose membrane (Protran).
RNA was recovered by digesting proteins using proteinase K and then reverse transcribed into cDNA. Reverse transcription primers and barcodes are indicated in the sample metadata. The cDNA was size selected (low – 70 to 85 nt, middle – 85 to 110 nt, high – 110 to 180 nt), circularized to add the adaptor to the 5' end, digested at the internal BamHI site, and then PCR amplified using AccuPrime SuperMix I (Life Technologies). The final PCR libraries were purified on PCR purification columns (Qiagen) to remove residual PCR reagents, and a ratio of 1:5:5 from the low, middle and high fractions were submitted for sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Extracted RNA was reverse transcribed using primer Rt10clip: /5Phos/NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGTCNN, where N are random bases.
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| Data processing |
CASAVA 1.8 was used for base calling.
Reads from two sequencing runs, one with 60 nt and one with 67 nt read length, were trimmed to 49 nt and combined. Reads consisted of 3 random positions, a 4-nt multiplexing barcode, and another 2 random positions, followed by the actual read. Duplicates were discarded.
Reads were de-multiplexed (note that they are already de-multiplexed in this GEO record), and the random positions, barcode, and 3'-bases matching Illumina adaptors were removed.
Remaining longer than 25 nt were mapped to the mouse transcriptome (Ensembl annotation of NCBIm37) using tophat with default settings. To prevent false assignments of reads from repetitive regions, any reads with a mapping quality < 3 were removed from further analysis.
Plots showing average crosslinking signal of events aligned to exon borders were generated as described previously for ChIP-seq data (Braunschweig et al., Genome Res (2014)), except that reads were first reduced to their first position, which is adjacent to the crosslink position, and no normalization against a control was performed. A 21-bp smoothing window average was used for display only.
Genome_build: mm9
Supplementary_files_format_and_content: PDF files contain figures showing the average (across represented exons) CLIP per million in the library, for each aligned base. Plots were generated directly from BAM files.
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| Submission date |
Dec 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ulrich Braunschweig |
| Organization name |
University of Toronto
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| Department |
Donnelly Centre
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| Lab |
Benjamin J. Blencowe
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| Street address |
160 College Street
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE80202 |
Multilayered control of alternative splicing regulatory networks by transcription factors (iCLIP-Seq) |
| GSE80205 |
Multilayered control of alternative splicing regulatory networks by transcription factors |
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| Relations |
| BioSample |
SAMN06131861 |
| SRA |
SRX2415968 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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