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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 01, 2012 |
| Title |
Barcode_B12_rna_46c_d5_nuc_pa_p1_60 |
| Sample type |
SRA |
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| Source name |
46C mESC cell line
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES-derived neural precursors (46C)
cell compartment: nucleus
fragmentation: Nuclease P1, 60 min
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| Treatment protocol |
Cells were fractionated to their nuclear and cytoplasmic components using a gentle detergent to disrupt the outer cell membrane while preserving intact nuclei before RNA extraction.
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| Growth protocol |
mESCs (cell line 46C) were cultured without feeder layers according the BayGenomics protocol adapted from (Skarnes WC, Methods Enzymol. 2000;328:592-615). mESCs were differentiated to Day 5 neural precursors with the protocol described by Ying et al. (Ying QL et al., Methods Enzymol. 2003;365:327-41; Ying QL et al., Nat Biotechnol. 2003 Feb;21(2):183-6). Briefly, undifferentiated ES cells are plated as a monolayer on gelatin-coated dishes in media lacking leukemia inhibitory factor (LIF), serum, and any growth factors; the absence of LIF prompts mESC differentiation, and in conditions without serum or growth factors the cells largely commit to the neural lineage. The cells acquire a neural precursor morphology by 5 days after plating.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Extracted RNAs (approximately 100-600 ug) were treated with DNase to remove contaminating DNA, then polyadenylated transcripts were selected with Oligotex resin (Invitrogen). We then chose to remove the polyA tails to allow sequencing up to the very 3' ends of the transcripts; to do this we annealed an oligo dT primer (Invitrogen) to the RNA samples, then treated with RNase H (New England Biolabs). We purified these RNAs using a G50 column (Roche) to remove oligos smaller than 25bp, followed by another DNase treatment, and then used the Ribominus kit (Invitrogen) to deplete our RNAs further of rRNA. Next, RNAs (400 ng) were fragmented using either 1 ug Nuclease P1 (Sigma) for 10 or 60 minutes or 1 U RNase III (Applied Biosystems, now Life Technologies) for 10 minutes, according to the instructions in the SOLiD Whole Transcriptome Analysis Kit (Applied Biosystems). The final libraries were generated from these fragmented RNAs using the Small RNA Expression Kit (SREK, Applied Biosystems), with insert sizes of approximately 90-190 bp. Equimolar concentrations of each library (excepting Barcode B06, for which we used twice the concentration) were used as the starting material for emulsion PCR.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD System 3.0 |
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| Data processing |
Reads were mapped in sequential stages to the mouse genome using the Applied Biosystems SOLiD Small RNA Analysis pipeline (Corona), as described previously (Underwood JG et al., Nat Methods. 2010 Dec;7(12):995-1001). In summary, we generated a filter file that included SOLiD adaptor sequence, rRNAs, and small RNAs (including tRNAs, miRNAs, and others). We also generated a target file (exon+introns) based on UCSC Known Genes: this file contains for each gene the complete genomic sequence from transcription start to transcription end; overlapping genes were merged. Finally, we generated a file containing known exon junction sequences; this file contains 50 bases upstream and downstream of all unique splices represented in UCSC Known Genes. To map reads, we first removed any reads mapping to the filter file, then mapped to the exon+introns target file (sense and antisense). Any remaining reads were then mapped to the genome. We then performed a second round of mapping, first filtering the reads as in the first round, and then mapping to the splice junction file. At each mapping stage, up to 15 hits per read were allowed. Any reads mapping more than 15 times were discarded. Mapping to the genome was performed chromosome by chromosome, and thus up to 15 hits per chromosome were allowed.
Sense and antisense mappings from the exon+introns target file were converted to positive and negative strand mappings to the genome. All read mappings for a given barcode were combined to one bed file.
Genome_build: mm9
Supplementary_files_format_and_content: All files in UCSC bed format.
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| Submission date |
Jun 28, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Courtney Onodera |
| E-mail(s) |
courtney.onodera@ucsf.edu
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| Organization name |
University of California, San Francisco
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| Street address |
1450 Third St, HD374
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL9318 |
| Series (1) |
| GSE38990 |
The polyA+ transcriptomes of mouse embryonic stem cells and their derived neural precursors from strand-specific RNA-Seq |
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| Relations |
| SRA |
SRX157483 |
| BioSample |
SAMN01085346 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM953368_Barcode_B12_rna_46c_d5_nuc_pa_p1_60_all_F3.50.6.trim.allMappings.mm9.bed.gz |
213.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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