|
| Status |
Public on Aug 04, 2014 |
| Title |
TH cells from LC, Mecp2 null, young time point, biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
brain, locus coeruleus neurons, Mecp2 null
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
pooled: No
amplification batch: Aug-11
# cells: 72
Sex: male
region: LC (locus coeruleus)
age: P22
genotype: Y/Mecp2-;TH/-
tissue: brain
cell type: tyrosine hydroxylase (TH)-positive locus coeruleus (LC) neurons
|
| Treatment protocol |
Mice at postnatal day 22-25 were sacrificed, the brain extracted and sliced on a vibratome (Leica). Slices were incubated in ACSF with pronase E (1mg/mL; Sigma) for 1 hour and desired regions were microdissected. Three Pasteur pipettes with decreasing tip size were used for tritration in a 1.5 ml Eppendorf tube. Tritrated samples were diluted 20 times with ACSF with FBS (1%) and plated on a 10cm petri dish with Sylgard substriatum. Glass pipette of tip size around 50 um was used to aspirate fluorescent cells under a fluorescent stereomicroscope (Leica). Aspirated cells were transferred to a new dish with fresh ACSF and aspirated again to purify the sample. This wash was repeated twice more (total of 3 washes). After the last wash, the sample was transfered to a 200 ul PCR tube with 50 ul of XB (extraction and lysis buffer from PicoPure RNA Isolation Kit; Arcturus).
|
| Growth protocol |
Mecp2 heterozygous null female mice were mated to male mice. Either male or female carried one of the fluorescent alleles (G42, YFPH or TH). Male offsprings which are hemizygous to Mecp2 null allele and hemizygous to one of the fluorescent alleles were used for experiments. Littermates which are wild type for X chromosome and hemyzygous to one of the fluorescent alleles were used for controls. Mice were weaned around P30. Normal 12 hour-12 hour light dark cycle were used. Normally only up to 4 mice were put in a cage.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PicoPure RNA Isolation Kit (Arcturus) was used to extract total RNA according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared using two rounds of T7 in vitro transcription based aRNA amplification (Affymetrix Small Sample Target Labeling Assay Version II) from about 0.3 to 1 ng of total RNA.
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| |
|
| Hybridization protocol |
Following fragmentation, 15 or 20 ug of cRNA were hybridized for 18 hr at 45C on GeneChip Mouse Genome 430 v2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned with the Affymetrix GeneArray Scanner G3000.
|
| Description |
Gene expression data from TH GFP labeled neurons from the locus coeruleus (LC), Mecp2 null
TH_LCY_KO_3
|
| Data processing |
The data were processed with fRMA (McCall MN, Bolstad BM, Irizarry RA (2010) Frozen robust multiarray analysis (fRMA). Biostatistics 11:242-253) algorithm in the Bioconductor with default settings.
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| |
|
| Submission date |
Aug 04, 2014 |
| Last update date |
Aug 04, 2014 |
| Contact name |
Ken Sugino |
| Organization name |
Janelia Research Campus
|
| Lab |
NeuroSeq
|
| Street address |
19700 Helix Dr
|
| City |
Ashburn |
| State/province |
VA |
| ZIP/Postal code |
20147 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE8720 |
Cell type specific expression data from Mecp2 null mice |
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