Protocols: Rat tibialis muscle samples not subjected to Laser capture microdissection (groups w/a, w/o, f/a):Snap frozen rat tibialis muscles were cut on a cryostat and 20 to 30 serial sections (10 mm) were collected in 2 ml tubes (Micro tube 2 ml PP, Sarstedt #72.694.006) with ceramic beads (Lysing matrix D bulk beads, MP #6540-434) precooled on dry ice.Rat tibialis muscle samples subjected to Laser capture microdissection (groups f/o+, f/o-):Skeletal muscles were rapidly dissected and mounted on cork with little optimal cutting temperature compound and snap frozen in liquid nitrogen. One third from the proximal tibialis muscle was cut off. (The first 15 μm cross-sections were used for the RNAscope® assay for Cdkn1a and Tmem158 as well as and for RNA integrity analysis of the tissue). Consecutive sections were placed on SuperFrost Plus™ adhesion slides (Thermo Scientific™), fixed in PAXgene Tissue FIX (Qiagen Cat No./ID: 765312), followed by dehydration steps in ethanol (50%, 70%, 90% 2 × 100% 1 min each), and allowed to air-dry. LCM was performed using a Zeiss PALM (Photo-Activated Localization Microscopy) microscope. Areas of interest were marked, guided by RNAscope® and the distinct shape of senescence/denervation marker positive muscle fibers. AutoLPC (automated laser pressure catapulting) was used to capture selected areas. Handling time for each sample was kept to 25 min. Sampling tubes were immediately put on dry ice with the lid upside down and transferred to −80 °C until RNA extraction. Rat tibialis muscle samples not subjected to Laser capture microdissection (groups w/a, w/o, f/a): For the phenol-chloroform based QIAzol procedure, muscle sections were lysed in 1000μl QIAZol (Qiagen #79306), homogenized for 10 sec at 6000 rpm using the Precyllis (Bertin Instruments #P000669-PR240-A) and proceeded following the manufacturer recommendation. The aqueous phase was transferred to a 2 ml Eppendorf tube. RNA was purified using a customized protocol for the Qiacube for large volume with DNase digest and the RNeasy Micro Kit (Qiagen #74004). RNA purity was determined using the Experion™ Automated Electrophoresis System. Rat tibialis muscle samples subjected to Laser capture microdissection (groups f/o+, f/o-): Total RNA was purified from the captured samples with the Arcturus® PicoPure® RNA isolation kit. TruSeq Stranded Total RNA Sample Preparation with Ribo-Zero Gold; average library insert size: 207 [196 - 235].
ENA first public
2021-06-26
ENA last update
2021-06-26
External Id
SAMEA9211519
INSDC center alias
Novartis Institutes for BioMedical Research
INSDC center name
Novartis Institutes for BioMedical Research
INSDC first public
2021-06-26T00:12:41Z
INSDC last update
2021-06-26T00:12:41Z
INSDC status
public
Submitter Id
E-MTAB-10582:AS_IL_LIB_17_01EL
age
15
broker name
ArrayExpress
common name
Norway rat
developmental stage
adult
genotype
wild type genotype
individual
84
material type
whole muscle
organism part
tibialis anterior
sample name
E-MTAB-10582:AS_IL_LIB_17_01EL
sex
male
strain
Sprague-Dawley