|
| Status |
Public on Nov 01, 2011 |
| Title |
RNA-Seq Sox2/Sox3 siRNA in neural progenitor cells, after 24h |
| Sample type |
SRA |
| |
|
| Source name |
Neural progenitor cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: ES-cell derived neural progenitor cells
differentiation: 4 days of differentiation
modification: Sox2 and Sox3 siRNA (Invitrogen) transfected, 24h
|
| Treatment protocol |
siRNA transfection was done by Lipofectamine treatment. Magnetic beads was used for PSA NCAM sorting.
|
| Growth protocol |
Mouse ES-cells were differentiated with B27 and N2 supplement (GIBCO) as monolayers on gelatinized cellbinding surface (Corning) with 0.5uM retinoic acid (all-trans, Sigma) at day 0 and day 2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Libraries were constructed from total RNA. Libraries were prepared according to Illumina's instructions accompanying the mRNA-Seq sample preparation kit (Part # 1004898 Rev.D). Briefly, the mRNA was purified from total RNA by PolyA selection. The mRNA was subjected to fragmentation using divalent cations under elevated temperature. cDNA synthesis was performed with fragmented RNA, and cDNA was end-repaired with a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3? to 5? exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3? end. After adapter ligation, size selection was achieved by excising a band of 200±25 bp from an agarose gel to remove excess adapters. The gel purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Sox2/Sox3 knockdown
library selection: Poly(A)-selection
|
| Data processing |
Reads were aligned by Bowtie, with options -n 2 -m 1, against genome build mm9.
Expression values (FPKM) were calculated by rpkmforgenes.py (http://sandberg.cmb.ki.se/rnaseq/) with options -mRNAnorm -fulltranscript and RefSeq gene annotation downloaded from UCSC genome browser 1 July 2010.
|
| |
|
| Submission date |
Oct 18, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Rickard Sandberg |
| E-mail(s) |
Rickard.Sandberg@ki.se
|
| Organization name |
Karolinska Institutet
|
| Department |
Department of Cell and Molecular Biology
|
| Street address |
Berzelius vag 35
|
| City |
Stockholm |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL11002 |
| Series (2) |
| GSE33024 |
Sequentially acting Sox transcription factors in neural lineage development |
| GSE33060 |
Sequentially acting Sox transcription factors in neural lineage development [RNA-seq] |
|
| Relations |
| SRA |
SRX101766 |
| BioSample |
SAMN00740004 |
