title: GSM4673234: NMJs from α-LTx treated mouse, collected 4 hours after α-LTx injection, replicate 1; Mus musculus; RNA-Seq
library_construction_protocol: After different time points (0, 4, 16, 72 and 168 h) treated mice (5 mice /group) were sacrificed by anaesthetic overdose followed by cervical dislocation, then soleus muscles were rapidly collected, and frozen in isopentane cooled with liquid nitrogen. Cryo-sections (7-10 µm thick) were transferred to UV-treated microscope glass slides. Microdissection was performed under direct microscopic visualization with PALM RoboMover automatic laser microdissector (Carl Zeiss, Oberkochen, Germany). One hundred NMJs/sample were collected by laser microdissection (LCM) within 30 min, to preserve RNA integrity, and pooled. Total RNA was isolated by incubation with 50 µl lysis buffer PKD (Qiagen) and 10 µl proteinase K (Promega) at 55°C o.n., with the sample upside down. The day after samples were centrifuged for 10 min at 10000 rpm, and RNA extracted with the automated system Maxwell 16 (Promega), using the Maxwell® 16 LEV RNA FFPE Purification Kit. RNA extraction was performed following manufacturer's protocol starting from the DNase treatment step. cDNA synthesis was obtained using the SMARTer Universal Low Input RNA kit (Clontech Laboratories), according to manufacturer's instructions. Libraries for RNA-Seq were obtained using the Nextera XT kit (Illumina) according to manufacturer's guidelines. RNA sequencing was performed with NextSeq 500 (Illumina), loading a maximum of 6 pooling libraries for each cartridge NextSeq High Output (300 cycles).
attributes.tag: GEO Accession
attributes.value: GSM4673234