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Pipeline publication

[…] rength of the mouse. Age and litter matched non-transgenic female mice served as control. At 95 days of age, mice were sacrificed by intraperitoneal injection with ketamine chlorohydrate (100 mg/kg) and xylazine (5 mg/kg) and intracardially perfused with PBS at 4°C. Lumbar spinal cords were quickly dissected, fresh frozen, and kept at −80°C until further analysis., RNA-sequencing was performed as previously described (Henriques et al., ). Briefly, total RNA was extracted from frozen samples of spinal cord from 95 days old mice (n = 5/group). Libraries of template molecules suitable for high throughput DNA sequencing were created and reads were mapped onto mm10 assembly of mouse genome using Tophat v2.0.14 (Kim et al., ). Quantification of gene expression was performed using HTSeq v0.6.1 (Anders and Huber, ) and Ensembl release 81 database. Supervised statistical analysis for differential gene expression has been performed using R (3.3.2) and the DESeq2 Bioconductor (v3.2) library. Multiple testing was adjusted by Benjamini and Hochberg FDR correction (Benjamini and Hochberg, )., After normalization and rlog transformation, hierarchical clustering (single method), and PCA (principal component analysis) was performed with the indicated subset of genes, accordingly to KEGG pathways, using JMP 11.0.0. Validation of gene expression was assessed by qPCR with a CFX96 using SYBR green Supermix reagent (BioRad), with the samples used for RNA-sequencing. Relative quantification of each gene was determined using the Biorad software and normalized to reference genes (Pol2, TBP, and 18S). Primers sequences are provided in Supplementary Table . C […]

Pipeline specifications

Software tools TopHat, HTSeq, DESeq2
Organisms Homo sapiens, Mus musculus
Diseases Motor Neuron Disease, Proteostasis Deficiencies, Spinal Cord Diseases, TDP-43 Proteinopathies, Motor Neuron Disease
Chemicals Phospholipids, Glycols, Amino Alcohols