|
| Status |
Public on Dec 31, 2017 |
| Title |
Hippocampal_neurons_4h_KCL_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
hippocampal neurons
|
| Organism |
Mus musculus |
| Characteristics |
tissue: cultured hippocampal neurons
differentiation state in vitro: 11 days
developmental stage: E18.5
mouse number: 31
strain: C57BL/6J
|
| Treatment protocol |
samples treated or untreated with 55mM KCl
|
| Growth protocol |
1000Xg, cells were resuspended in complete NB medium containing 22µM L-glutamic acid (Sigma, Germany), counted, and seeded on poly-ornithine (0.1 mg/ml, Sigma) and laminin (1μg/ml, Sigma) coated plates. Medium change was performed every 96h by exchanging half of the volume of the medium with fresh NB medium with complements and AraC (1µg/ml for the first, and 0.5µg/ml for the second medium change, Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
C57BL/6J (Janvier Labs, France) hippocampi of E18.5 embryos were dissected and collected in 15 ml Hanks’ Balanced Salt Solution (HBSS, PAA, Germany). After centrifugation (5min; 800xg) hippocampi were dissociated by adding 1ml 0.05% trypsin-ethylenediaminetetracetic acid (PAA) at 37°C for 15min. After adding 1ml of fetal bovine serum (FBS, Life Technologies) and 1ml of neurobasal (NB) medium supplemented as previously described (Vezzali R et al. 2016) samples were homogenized by repeated passages through a pipette tip. After 10min centrifugation at 1000Xg, cells were resuspended in complete NB medium containing 22µM L-glutamic acid (Sigma, Germany), counted, and seeded on poly-ornithine (0.1 mg/ml, Sigma) and laminin (1μg/ml, Sigma) coated plates. Medium change was performed every 96h by exchanging half of the volume of the medium with fresh NB medium with complements and AraC (1µg/ml for the first, and 0.5µg/ml for the second medium change, Sigma). At day in vitro (DIV) 11 cells were treated with 55mM KCl at 37ºC, 5%CO2 at different time points before harvesting.
|
| Label |
Biotin
|
| Label protocol |
We used the Affymetrix WT Plus kit for labeling as described by the manufacturer.
|
| |
|
| Hybridization protocol |
Labeled targets were hybridised to the arrays according to the manufacturers protocol (Affymetrix). Arrays were washed and stained using the Affymetrix Hyb and Stain kit
|
| Scan protocol |
Arrays were scanned using the Affymetrix Scanner 3000 7G and CEL files were processed using Command Console software (Affymetrix)
|
| Description |
Hippocampal neurons were isolated at day E18.5, cultured for 11 days in vitro and then treated with or without 55mM KCL, then the total RNA was isolated
|
| Data processing |
We used Partek Genomics Suite software for further analysis: Pre-background Adjustment was done for GC Content and probe sequence. We used RMA background correction and Quantile normalization. Probes were log2 transformed and probeset summarization was done with median polish. Probe values were log2 transformed.
|
| |
|
| Submission date |
Nov 25, 2016 |
| Last update date |
Dec 31, 2017 |
| Contact name |
Henriette Franz |
| Organization name |
University Freiburg
|
| Department |
Anatomy and Cellbiology
|
| Lab |
Vogel
|
| Street address |
Alberstr: 17
|
| City |
Freiburg |
| ZIP/Postal code |
79104 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16570 |
| Series (2) |
| GSE90508 |
Neuronal activity, TGFβ signaling and unpredictable chronic stress affect transcription of Gadd45 family members in vitro and in vivo [Affymetrix] |
| GSE90513 |
Neuronal activity, TGFβ signaling and unpredictable chronic stress affect transcription of Gadd45 family members in vitro and in vivo |
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