NM0204_UN_Homo4
Mus musculus
Protocols: Schwann cell cultures were prepared from sciatic nerves essentially as in Morgan et al., (1991) and Arthur-Farraj et al., (2011). After enzymatic dissociation and centrifugation, the cell pellet was resuspended in defined medium (DM) (Meier et al., 1999), containing 10 -6 M insulin and 5% HS, and plated in drops on coverslips coated with Poly-L-lysine and laminin. Cells were incubated at 37°C/5%CO2 and allowed to adhere for 24 hr. After 24 hr, the medium was changed to DM/0.5% HS (controls), or DM with 10ng/ml NRG1 alone, or DM with NRG1 10ng/ml and dbcAMP 1mM for 48 hr before fixation and immunolabelling. Total RNA was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen) with a DNase I step performed to eliminate traces of genomic DNA. The purified mRNA was fragmented, and primed with random hexamers. Strand-specific first strand cDNA was generated using Reverse Transcriptase in the presence of Actinomycin D. The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. The resultant cDNA was then “A-tailed” at the 3' end to prevent self-ligation and adapter dimerisation. Truncated adaptors, containing a T overhang were ligated to the A-Tailed cDNA. Successfully ligated cDNA molecules were then enriched with limited cycle PCR (10-14 cycles).
ENA first public
2017-10-16
ENA last update
2017-10-13
External Id
SAMEA104349050
INSDC center alias
The UCL Institute of Child Health
INSDC center name
The UCL Institute of Child Health
INSDC first public
2017-10-16T17:02:18Z
INSDC last update
2017-10-13T14:55:40Z
INSDC status
public
Submitter Id
E-MTAB-6138:NM0204_UN_Homo4
broker name
ArrayExpress
cell type
Schwann cell
common name
house mouse
developmental stage
adult
genotype
P0-Cre+;R26c-Junstopff/f
organism part
sciatic nerve
phenotype
28 fold c-Jun overexpression
sample name
E-MTAB-6138:NM0204_UN_Homo4
sex
male
strain
C57BL/6