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| Status |
Public on Jun 12, 2013 |
| Title |
Upf1.CHX.A2_CLIPSeq |
| Sample type |
SRA |
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| Source name |
CLIP-Seq analysis of Upf1
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| Organism |
Mus musculus |
| Characteristics |
cell line: v6.5
antibody: rabbit anti-Upf1 (Bethyl Labs Cat. No. A301-902A)
cell type: Embryonic stem cells
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| Treatment protocol |
For CHX treatment, 100ug/ml cycloheximide was added to culture media for 2hours at 37deg prior to harvest.
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| Growth protocol |
mESCs were cultured on MEFs in standard mESC medium with LIF at 37deg, 5% CO2. For shRNA-expressing clones, 1.5ug/ml puromycin was added to media. Prior to any RNA isolation technique, mESCs were pre-plated for 30minutes on gelatinized culture dishes to remove MEFs. Only mESCs remianing in suspension were collected for analysis. For shRNA-expressing clones, puromycin was removed from media 48hours prior to analysis. For CLIP-Seq libraries, mESCs were pre-plated to remove MEFs and plated off of MEFs 24 hours in gelatinized culture dishes prior to UV irradiation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For CLIP-Seq libraries, mESCs were UV irradiated (254 nm, 400mJ/cm2) in 4ml PBS on ice and then trypsinized, washed with PBS, and snap frozen in liquid nitrogen. Frozen cell pellets were lysed directly in lysis buffer (see Methods).
CLIP-Seq libraries were prepared essentially as described in Wang et al. 2012 that added 3' and 5' adapters by RNA ligation. 5' adapter added 2 randomized nucleotides to enable filtering of reads based on PCR amplificaiton.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
small RNA fragments bound to Upf1
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| Data processing |
Library strategy: CLIP-Seq
Paired-end mRNA-Seq and ribosome footprinting libraries were mapped to the mouse genome (mm9) with a splice junction database using Tophat v.1.4.0 (Trapnell et al. 2009) allowing 2 mismatches but disallowing splice site mismatches and novel intron discovery (--solexa1.3-quals --splice-mismatches 0 --min-intron-length 10 --max-intron-length 1000000 --min-isoform-fraction 0.0 --no-novel-juncs). Isoform and gene expression values were then estimated using Cufflinks v.1.3.0 (Trapnell et al. 2010) and a combined Ensembl and UCSC annotation database of predicted coding genes (with duplicate isoforms removed) (--compatible-hits-norm). To maximize detection of lowly expressed isoforms, mapped files from like shRNA hairpins were merged before gene expression quantitation. For mRNA-Seq libraries, mapping to the entire mature mRNA sequence was used to estimate isoform and gene expression values. For ribosome footprinting libraries, mapping to only the coding sequence was used to estimate gene expression values.
CLIP-Seq libraries were filtered for unique sequences, trimmed for remaining adapters and 2 nucleotide randomized barcode, and mapped uniquely to the mouse genome (mm9) and a database of splice junction sequences using Bowtie v.0.12.6 (Langmead et al. 2009) allowing 2 nucleotide mismatches (-m 1 --best –strata). Reads from each IgG library were amplified by 10x and then subtracted from their experimentally matched Upf1 library by iteratively cancelling out reads that overlapped. Barcodes which were likely (p >= 0.05) misreads of other highly amplified barcoded reads at the same position were then predicted based on a Poisson model and removed.
Genome_build: mm9
Supplementary_files_format_and_content: For mRNA-Seq and ribosome footprinting libraries, isoform and gene expression values (output of Cufflinks software) are provided.
Supplementary_files_format_and_content: For CLIP-Seq libraries, a bed file describing read positions is provided for each Upf1 sample. This file has been filtered for overlapping IgG reads and "one-off" barcodes that were predicted to be due to PCR amplification. Note that for determiniation of gene binding, Upf1 positions were further collapsed for unique barcode/position combinations.
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| Submission date |
Oct 23, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Jessica Hurt |
| E-mail(s) |
[email protected]
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| Organization name |
Massachusetts Institute of Technology
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| Department |
Biology
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| Lab |
Christopher Burge
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| Street address |
77 Massachusetts Avenue
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE41785 |
Global analysis of Upf1 in mESCs reveals expanded scope of nonsense-mediated mRNA decay |
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| Relations |
| SRA |
SRX200666 |
| BioSample |
SAMN01779623 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1024305_Upf1.CHX.A2_sub_barcodeFilter_CLIPseq.bed.gz |
517.9 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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