Nucleospin rna plus xs

Bulk sequencing of tetramer-positive cells (SMARTer Human TCR a/b Profiling Kit v2, Takara Bio USA) was performed after amplifying cDNA from mRNA (NucleoSpin RNA Plus XS, Takara Bio USA), using an Illumina MiSeq system (paired-end 300 bp). Raw reads were assembled using Cogent NGS Immune Profiler Software (Takara Bio USA).

Two mm SC sections, including the lesion site plus additional rostral and caudal tissue proximal to the lesion, were collected for qRT-PCR. Total RNA was prepared using NucleoSpin RNA Plus XS (Clontech, cat# 740990) and cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit (ThermoFisher, cat# K1672) according to manufacturer’s specifications. Quantitative PCR was completed using the Luna polymerase master mix (NEB, cat# M3003) using gene-specific primers (Table S1). Primers were designed to flank introns and were confirmed to not amplify project from genomic DNA. To determine primer efficiency, a standard curve was generated for each primer set using cDNA pooled from wild-type embryos at 1, 3, and 5 days post-fertilization. qRT-PCR was performed on a Bio-Rad CFX Connect Real-Time System. For each gene, log2(fold change) was calculated using the DCq method and normalized to eif1a as a loading control and to control gene expression for each experiment.

Two mm SC sections, including the lesion site plus additional rostral and caudal tissue proximal to the lesion, were collected from mstnb mutants and wild-type siblings at 7 dpi. Uninjured mstnb mutants and wild-type SCs were also collected. Total RNA was prepared using NucleoSpin RNA Plus XS (Clontech, cat# 740990) and sent for bulk RNA sequencing. TruSeq libraries were prepared and sequenced on Illumina HiSeq 3000 using 50 bp single-end reading strategy. Quality QC and trimming of adapters and short sequences were performed using Fastx. Sequencing reads were mapped to the zebrafish genome (Zv11) using Bowtie2, then assembled and quantified using the Cufflinks and Cuffdiff algorithms. Genes with log₂(fold enrichment) between −1 and 1 or adjusted p-value ≥ 0.01 were considered insignificant. RNA sequencing was performed at the Genome Technology Access Center at Washington University. Analysis was performed in the Bioinformatics Core at the Center for Regenerative Medicine at Washington University. This dataset is available at GEO (accession number: GSE183644).
RNA-seq data (GEO accession number: GSE77025) was used to evaluate the expression of Tgf-β ligands after complete SC transection. Log₂(fold change) is expressed for SCs at 7 dpi relative to the sham injured SCs.^{15 (link)}