title: GSM8036311: 20m_KO_6; Mus musculus; RNA-Seq
library_construction_protocol: Motor neuron laser-microdissection was performed as previously described (Lobsiger et al., 2007). Briefly, lumbar spinal cords were rapidly recovered, rinsed and frozen in -40°C Isopentane in HistoPrep OCT compound (Fisher Scientific International), cryosectioned (16 μm) and sections mounted on RNase-free PENfoil covered glass slides (Leica Microsystems). Sections were fixed for 60 s in 75% EtOH and stained for 30 s in 4% cresyl-violet acetate/0.3% acetic-acid (Sigma) to identify motor neurons, dehydrated in graded solutions of ethanol (70%, 95%, 100%) and dehydrated for 1 h before laser-microdissection (Leica LMD-7000). From one spinal cord, 200 large (diameter ≥ 25 μm) ventral horn motor neurons of the lumbar L4–L6 region were collected dryly, then immediately lysed in lysis buffer (NucleoSpin RNA XS Kit, Machery-Nagel) and the lysate stored at -80°C. Approximately 10 ng of total RNA can be recovered per 200 laser-microdissected motor neurons (NucleoSpin RNA XS Kit; with DNase treatment) Since the RNAconcentration was too low to be detected using a Nanodrop, test samples were used to assess  RIN quality. Since the same number of motor neurons were taken per sample, the same volume of RNA was used (10µL).cDNA synthesis was performed using the Smartseq V4 TAKARA kit with linear pre-amplification. Library preparation was made with amplified cDNA for every sample using the Nextera XT DNA Library Preparation Kit (Illumina). 47 samples were run with a 1600 million cartridge to reach an average coverage of 34 million reads per sample (100bp, paired-ends). Sequencing was performed using the Novaseq 6000 sequencer (Illumina). Library preparations and sequencing were performed by the ICM iGenSeq core facility.