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| Status |
Public on Dec 29, 2012 |
| Title |
plusRA_RNA-seq |
| Sample type |
SRA |
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| Source name |
NTera2/D1 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: NTera2/D1
treatment: RA
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| Treatment protocol |
For induction, the cells were treated with 1 μM atRA (Sigma) for 2 days.
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| Growth protocol |
Ntera2/D1 cells (American Type Culture Collection) were kept in minimum essential medium α (Invitrogen) plus 10% FBS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from untreated and RA-treated NTera2 cells using the Trizol reagent (Invitrogen) and reverse-transcribed with SuperScript III kit (Invitrogen).
10 µg total RNAs were subjected to magnetic oligo(dT) beads for selecting poly(A)-tailed RNAs. The resultant RNA samples were then fragmented into small pieces by heating to 94°C, followed by EtOH precipitation. The fragmented RNAs were reverse-transcribed to the first strand of cDNAs by using the Superscript III kit (Invitrogen). The second strand of cDNAs was then synthesized by RNA Pol I. The double-stranded cDNAs were end-repaired by T4 DNA polymerase and Klenow polymerase. The blunt-ended double-stranded cDNAs were added an "A" base by Klenow fragment. The adaptors were added to the double-stranded cDNAs by the T4 DNA ligase. By separating the sample on 2% agarose gel, gel slice around 200 bp was cut out and subjected to gel extraction. The extracted cDNAs were amplified by PCR to obtain the library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
The image analysis and base calling were performed by using Illumina's Genome Analysis pipeline.
To quantify the gene expression, the counts/RPKM values for were computed by using SeqMap and rSeq
To identify the Alu elements with a DR2 sequence motif, Alu sequences were scanned with a DR2 position weighted matrix (PWM) (PAZAR database) using PATSER at distinct mapping scores. The potentially induced DR2 Alu elements were identified on the basis of the difference of normalized read counts (–atRA compared to +atRA). To quantify the DR2 Alu expression levels, the 72-nt sequencing reads were aligned to the hg18 assembly using Bowtie. Both uniquely aligned reads and those aligned to less than 10, 100, 1,000 or 10,000 locations were considered in the analysis. For reads aligned to multiple regions, a single location was chosen on the basis of a Gibbs sampling algorithm using five or ten iterations. The sequencing reads count on DR2 Alu was computed using intersectBed tool in BedTools and normalized to the sequencing depth. Globally, the induction of DR2-Alu was assessed by Z-test.
Genome_build: hg18
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each sample, and a list of potentially induced DR2-Alu
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| Submission date |
Nov 27, 2012 |
| Last update date |
Jun 11, 2013 |
| Contact name |
MICHAEL G ROSENFELD |
| E-mail(s) |
mrosenfeld@ucsd.edu
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| Organization name |
HHMI/UCSD
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (2) |
| GSE42563 |
DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation (RNA-seq) |
| GSE42602 |
DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation |
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| Relations |
| BioSample |
SAMN02197189 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1045173_induced.DR2-Alu.txt.gz |
104.3 Kb |
(ftp)(http) |
TXT |
| GSM1045173_plusRA.rseq.txt.gz |
417.2 Kb |
(ftp)(http) |
TXT |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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