Illumina HiSeq 4000 paired end sequencing; RNA sequencing of corticospinal neurons and control callosal projection neurons purified from the cerebral cortex of adult wild-type and Sod1G86R mice, a model of amyotrophic lateral sclerosis, at four disease-relevant ages.
RNA sequencing of corticospinal neurons and control callosal projection neurons purified from the cerebral cortex of adult wild-type and Sod1G86R mice, a model of amyotrophic lateral sclerosis, at four disease-relevant ages.
CSN from Sod1G86R mice and their wild-type littermates were retrogradelly labelled using Green IX Retrobeads (Lumafluor) injected at 25 days of age. Purification of CSN and CPN were performed in parallel from same WT or Sod1G86R mice at 30, 60, 90 and 105 days of age. Mice were deeply anesthetized with by an i.p. injection of Ketamine/Xylazine (100 mg/kg; 5 mg/kg) before decapitation. The brains were sectioned in a stainless steel coronal brain matrix (Harvard Apparatus, MA), and 1 mm-thick sections were transferred under a fluorescence SMZ18 microscope (Nikon). The layer V containing labelled CSN and the upper layer II/III above were microdissected from 4 coronal sections and collected in separate tubes filled with iced HABG (Hibernate A (BrainBits UK), B27, Glutamax (Gibco) and 0.1N NaOH ). The microdissected tissues were enzymatically digested with 34 U/ml papain at 37°C for 30 min. Cells were mechanically dissociated by gentle trituration in iced HABG, filtered through 70 μm cell strainer (BD Falcon) and subjected to density centrifugation through a three-density step gradient of Percoll (Sigma, MO) as previously described (Guez-Barber et al, 2012). Upon centrifugation, the cell pellets were resuspended in cold PBS and fixed with 70% ethanol for 30 min at 4°C. Fixed cells were centrifugated to eliminate EtOH and resuspended in 0.1 M PBS complemented with RNAse inhibitors (Promega). Microsphere-labelled CSN were purified using the FACS AriaII (BD Biosciences), based on their fluorescence, size and granularity. Unlabelled CPN were purified by FACS, based on their size and granularity. Approximately 2 000 CSN and exactly 2 000 CPN were be obtained from each adult mouse brain and were used as individual biological replicates for RNA sequencing. Upon purification, neurons were centrifugated and frozen as cell pellets.  Full length cDNA were generated from frozen cell pellets using the SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (Clontech, CA) according to the manufacturer's instructions. 11 cycles of cDNA amplification were performed using the Seq-Amp polymerase. 600 ng of pre-amplified cDNA were used as input for Tn5 transposon tagmentation by the Nextera XT kit (Illumina) followed by 12 cycles of library amplification. Following purification using Agencourt AMPure XP beads (Beckman Coulter), the size and concentration of the libraries were assessed on an Agilent 2100 Bioanalyzer.