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| Status |
Public on Jun 30, 2015 |
| Title |
LCL19_Polysome |
| Sample type |
SRA |
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| Source name |
Coriell lymphoblast cell line (LCL)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LCL19
fraction: polysome
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| Biomaterial provider |
Coriell Institute for Medical Research
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| Growth protocol |
cells were grown to 5x105 cells/mL density in T75 tissue culture flask and harvested.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with Trizol reagent from the cytoplasm of the three LCLs (total cytosol content), and from pooled polysome sucrose fractions (>3 ribosomes; 250 µL). Two-step column purification and size separation were performed on each RNA sample. First, long RNA (>200 bases) was separated using SpinSmart RNA Purification column (Denville), and the flowthrough (<200 bases) was placed on a second column for small RNA (microRNA) separation with mirPremier microRNA isolation kit (Sigma-Aldrich, St. Louis, MO). RNA quality was evaluated on a Bioanalyzer.
Cytoplasmic (total) and polysome derived long RNA (25 ng each) was then converted to cDNA using the NuGen Ovation RNA-Seq System V2 (NuGen Technologies, San Carlos, CA), which uses both random hexamers and oligo-dT to amplify all RNA sequences, while suppressing cDNA formation from ribosomal RNA by >90%. ERCC RNA (External RNA Control Consortium, Ambion) controls were spiked into RNA prior to NuGen cDNA synthesis. The NuGen Ovation RNA-Seq kit produces non-stranded cDNA (3-5 microgram, measured with Qubit (Life Technologies, Foster City, CA)). The double-stranded cDNA derived from long RNAs was sheared to 150-200 bp fragments with a Covaris focused-ultrasonicator (Covaris, Inc. Woburn, MA) and recovered by centrifuging over an YM-30 spin filter (Amicon EMD Millipore Billerica, MA). Fragments longer than 100 bp were retained and eluted from the membrane.
We generated barcoded sequencing libraries from 100 ng of sheared cDNA using the NEBNext Fast DNA Library Prep Set for Ion Torrent sequencing (New England Biolabs, NEB, Ipswich, MA), as described [22,48]. In a separate step, purified small RNA fractions (containing 100 ng RNA) were used for library construction using Ion Total RNA-Seq Kit v2 for small RNAs (Life Technologies, Foster City, CA). Pooled barcoded RNA libraries were sequenced on the Ion Torrent Proton platform (Life Technologies, Foster City, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
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| Description |
RNA-Seq, ncRNA-Seq
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| Data processing |
aligned reads >25 bases in length to hg19 genome reference first with tophat under default parameters, then aligning the unmapped reads using a local and very sensitive approach with bowtie
expression estimated with cufflinks v.2.1.1 for gencode v18 and lncipedia annotation for isoforms and gene. Whole gene expression calculated as sum of isoform with "OK" status
SNPs called using default conditions of mpileup with samtools and placed within UCSC annotated genes using annovar. AEI calculated by combining SNPs called as heterozygous in both total and polysome of a sample across a gene
Genome_build: hg19
Supplementary_files_format_and_content: tab delimited text files with FPKM for each annotated isoform for each sample and vcf formatted SNP calls for each sample
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| Submission date |
Feb 20, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Amy Hite |
| Organization name |
The Ohio State University
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| Department |
Biomedical Informatics
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| Street address |
1800 Cannon Drive 250 Lincoln Tower
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| City |
Columbus |
| State/province |
OH - Ohio |
| ZIP/Postal code |
43210 |
| Country |
USA |
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| Platform ID |
GPL17303 |
| Series (1) |
| GSE66135 |
Allele-selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-coding and Noncoding RNAs, and RNA Isoforms |
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| Relations |
| BioSample |
SAMN03365937 |
| SRA |
SRX885707 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1614898_LCL19_P_SNPcalls.txt.gz |
5.4 Mb |
(ftp)(http) |
TXT |
| GSM1614898_LCL19_P_isoform.txt.gz |
6.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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