|
| Status |
Public on Nov 04, 2015 |
| Title |
knockdown control (shRNA against luciferase) repeat2 |
| Sample type |
SRA |
| |
|
| Source name |
cerebral cortex
|
| Organism |
Mus musculus |
| Characteristics |
cell line: CD-1
tissue: cerebral cortex
cell type: Differentiated NSC
|
| Treatment protocol |
Neural stem cells were transduced with recombinant lentivirus after 24 h of culture on poly-d-lysine pre-coated plates. Non-infected cells were eliminated using fresh culture medium containing 0.5 mg/ml of puromycin 1 day after infection. Infected cells were proliferated for 3-4 days and then were differentiated into OPCs for 3 days in DMEM/F12 medium containing Glutamax, non-essential amino acids, BSA, B27, N2 supplement, 20 ng/ml CNTF, and 40 ng/ml T3.
|
| Growth protocol |
Neural stem cells were cultured and passaged every 2 days as monolayers in MEM/F12 medium containing Glutamax, non-essential amino acids, B27, N2 supplement, 20 ng/ml EGF, and 20 ng/ml FGF. Cells were dissociated using Accutase (Invitrogen) and seeded onto poly-d-lysine-coated plates or dishes.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using the miRNeasy kit (Qiagen) under manufactor’s protocols. The quality was accessed by Bioanalyzer. Samples with high RNA integrity number (RIN) (>8) were used for library construction. 100ng total RNAs were used for each sequencing library.
RNA-Seq was performed on the polyadenylated fraction of RNA isolated from differentiated cell samples. Two biological replicates were used. 100 ng total RNAs were used for each sequencing library. RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).The samples were then sequenced using the Illumina HiSeq 2000 sequencer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads were mapped to reference genome (version mm9) using Tophat (version 1.3.3) which uses the Bowtie (version 0.12.7)read mapper, Tophat was fed with the option of“–no-novel-juncs”and option "–G" which supplies Tophat with known transcript mm9 GTF file (download from cufflinks website).
Mapped reads were converted to sam files with SAMTOOLS (version 0.1.18) and raw counts were calculated by HTSeq (version 0.6.1)
DESeq was used for differential gene expression analysis caused by lnc-OPC knockdown. The output from Deseq was filtered using the following more stringent criteria: (1) padj<0.02; (2) foldChange<0.5 (3) foldChange>2.
GO functional term enrichment analysis of differentially expressed protein coding genes was performed by using DAVID functional annotation.
Genome_build: mm9
differentially expressed protein coding genes identified by DESeq and DAVID GO term enrichment analysis
Supplementary_files_format_and_content: raw counts
|
| |
|
| Submission date |
Nov 03, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiaomin Dong |
| E-mail(s) |
xiaomin.dong@yahoo.com
|
| Organization name |
UT health
|
| Department |
Department of neurosurgery
|
| Lab |
Dr.Jiaqian Wu
|
| Street address |
1825 Pressler Street
|
| City |
houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE74647 |
Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination (RNA-Seq of differentiated NSC after lnc-OPC knockdown) |
| GSE74648 |
Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination |
|
| Relations |
| BioSample |
SAMN04233141 |
| SRA |
SRX1411159 |
