|
| Status |
Public on Mar 02, 2023 |
| Title |
13h post ICSI single zygote injected Morpholino Oct4A-replicate-09 |
| Sample type |
RNA |
| |
|
| Source name |
single cell whole transcriptome amplification
|
| Organism |
Mus musculus |
| Characteristics |
developmental stage: 13h post ICSI single zygote
injected: Morpholino Oct4A
strain: B6D2F1
|
| Treatment protocol |
ICSI using sperm from 8 to 12 weeks old B6D2F1 males was essentially as previsously described (Suzuki et al., 2010).
|
| Growth protocol |
The Oocytes incubated in kalium simplex optimized medium under mineral oil in humidified 5% CO2 (v/v air) at 37°C, until required (Yoshida et al, 2007).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
It was based on isolation of the cellular mRNA by oligo-dT beads, cDNA synthesis using random octamer and oligo-dT primers, poly-dG tailing, and PCR amplification using a single primer under very stringent conditions adequate for CG-rich sequences. The solid-phase capturing of the mRNA enabled depleting the cDNA from free cDNA synthesis pri- mers and abundant dNTPs thus avoiding the subsequent amplification of tailed primers or inefficient tailing. Together with the introduction of random primers (resulting in a fragmentation of the cDNA to a size amplifiable by PCR) the protocol therefore allowed for optimal conditions of all enzymatic reactions such as high concentrations of dNTPs, primer and enzyme, all of which were individually shown to contribute to increased sensitivity. The highest increase of sensitivity (100-fold) over the original Brady procedure was achieved by the use of poly-G tailing instead of poly-A tailing and the subsequent use of a single poly-C-containing primer. Our methods were based on (Hartmann and Klein, 2006) and further improved to apply for extraction of total RNA from mouse single cells.
|
| Label |
biotin
|
| Label protocol |
Primary cDNA amplification products were labelled in the presence of 3% formamide, 2.4 mM CP2-BGL primer (TCA-GAATTCATGCCGCCCCCCCGGCCC), dNTPs (0.35 mM dATP and dGTP, 0.3 mM dTTP and dCTP) and 0.05 mM labelled nucleotides. Sample cDNA was labelled with digoxygenin-dUTP (Roche) and aminodigoxygenin-dCTP (PerkinElmer, Rodgau-Ju ̈ gesheim), reference cDNA with biotin-dUTP (Roche) and biotin-dCTP (Invitrogen).
|
| |
|
| Hybridization protocol |
Corning, Schiphol-Rijk, Netherlands) were prehybridized with 5xSSC + 0.1% SDS + 0.1% BSA at 42C and hybridized in an Arraybooster hybstation (Implen, Munich) at 42C overnight. Washing steps following hybridization were at 50C twice in 1· SSC + 0.1% SDS for 10 min, twice in 0.5x SSC + 0.1% SDS for 10 min, and twice in 0.1x SSC for 30 min. Unspecific binding of labelled proteins was blocked with 1% blocking reagent for nucleic acid hybridization (Roche) followed by a staining procedure with anti-Dig-Cy5 and Streptavidin-Cy3 (each 16 mg/ml, Jackson Laboratories). Excess antibody/streptavidin was removed with 4x SSC + 0.2% Tween-20 and slides were scanned on a Genepix 4000A scanner (Axon Instruments, Union City). Experiments shown in Table 2 were performed using human cells hybridized to Human Genome OpArrays version 4. Protocol details are available upon request.
|
| Scan protocol |
Slides were scanned on GenePix 4000A scanner Axon Instruments, Union City). Numerical readout of fluorescence intensities (GPR file) was generated using GenePixPro 7 (Molecular Devices)
|
| Description |
Morpholino oct4-09
|
| Data processing |
background correction and normalization using limma package in R
|
| |
|
| Submission date |
Jan 05, 2015 |
| Last update date |
Mar 02, 2023 |
| Contact name |
Xin Lu |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Regensburg
|
| Street address |
Universitätsstraße 31
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE64648 |
Genome activity during the emergence of mouse embryonic totipotency (morpholino) |
| GSE64650 |
Genome activity during the emergence of mouse embryonic totipotency |
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