|
| Status |
Public on Jun 12, 2014 |
| Title |
SP1KO Flk+ replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
from differentiation of ES cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Flk+
strain: C57BL/6
genotype: Sp1 -/- knock-out
|
| Growth protocol |
ES cells were trypsinised and allowed to form embryoid bodies by plating in base methylcellulose (Stem Cell Technologies M3134) supplemented with 10% FCS, 100 units/ml Penicillin and 100 µg/ml Streptomycin, 1 mM glutamine, 0.15 mM MTG, 10 g/ml insulin (Sigma), 5% IL-3 conditioned media, 10 ng/ml recombinant mouse M-CSF (R&D Systems), 100 units/ml IL-1 (Peprotech) at a cell concentration previously determined to give similar numbers of EB. After at least 14 days EB were counted and assessed for the number of EB.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cells using TRIzol® (Invitrogen) according to manufacturer’s instruction. First strand cDNA synthesis was carried out using Superscript II (Invitrogen) according to manufacturer’s instructions using 250-500 ng of RNA. Real Time PCR was carried out using ABI SYBR® green master mix with 5 µl of diluted cDNA and 0.25 µM primer per 15 µl reaction on an ABI 7900HT machine.
|
| Label |
Cy3
|
| Label protocol |
25ng of each source sample RNA was labelled with Cy3 dye as per the protocol detailed in the Low Input Quick Amp Labelling Kit (Agilent Technologies – 5190-2305). A Specific Activity of greater than 6.0 was confirmed by measurement of 260 nm and 550 nm wavelengths with a NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Hybridization samples were prepared for a 8-pack microarray using 600 ng cRNA each according to the hybridization protocol from Agilent: One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling), loaded onto the 8-pack array slide and hybridized at 65 °C overnight
|
| Scan protocol |
After washing the microarray was scanned on an Agilent C Scanner using the Profile AgilentG3_GX_1Color for 8x60K microarrays (Dye channel: Green; Scan region: Scan Area (61 x 21.6 mm); Scan resolution (μm): 3; Tiff: 20 bit)
|
| Description |
SP1_KO_Flk_24
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) (protocol GE1_107_Sep09, Grid: 028005_D_F_20100614 and platform Agilent SurePrint G3 Mouse GE 8x60K). The raw data output by Feature Extraction Software was analysed using the LIMMA R package with quantile normalization and background subtraction.
|
| |
|
| Submission date |
Nov 19, 2013 |
| Last update date |
Jun 12, 2014 |
| Contact name |
Salam Adli Assi |
| E-mail(s) |
s.a.assi@bham.ac.uk
|
| Organization name |
University of Birmingham
|
| Department |
Institute for Cancer and Genomic Sciences
|
| Street address |
IBR
|
| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13912 |
| Series (2) |
| GSE52497 |
A crucial role of the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specification [array] |
| GSE52499 |
A crucial role of the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specification |
|
