title: GSM7777788: SCS 20Hz, replicate 3, scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Spinal cord motoneurons of mice in the sham group (n = 3 mice), untrained group (n = 3 mice), 10-20 Hz MS group (n = 3 mice),  10-20 Hz SCS group (n = 3 mice), 10-20 Hz DES group (n = 3 mice), 1-40 Hz DES group (n = 3 mice) were labeled with TRDA and sacrificed 4 weeks after SCI.  The freshly isolated spinal cord was sliced on a Vibratome (Leica, Germany) into 200- to 300-μm-thick coronal sections.  Fluorescence-labeled motoneurons were visualized under a fluorescence microscope (Zeiss) using a miniature operating system (Eppendorf, Germany), and the motoneurons were aspirated by glass electrodes and placed in preservation solution (Invitrogen, USA).  TRDA-labeled neurons were individually homogenized in 1 ml TRIzol (Invitrogen), and RNA was extracted with a RNeasy Mini Kit (Qiagen) or equivalent.  Total RNA (1 μg) was used to synthesize double-stranded cDNA. First, reference genome sequences and gene model annotation files of related species were downloaded from genome websites such as UCSC, NCBI, and ENSEMBL. Second, Hisat2 software (v2.0.1, http://daehwankimlab.github.io/hisat2/) was used to index the reference genome sequence. Finally, clean data were aligned to the reference genome via Hisat2 software (v2.0.1). Then, transcripts in FASTA format were converted from known gff annotation files and indexed properly. Then, with the file that served as the reference genome file, HTSeq (v0.6.1, https://htseq.readthedocs.io/en/master/) was used to estimate gene and isoform expression levels from the paired-end clean data. Differential expression analysis was performed using the DESeq2 Bioconductor package, a model based on a negative binomial distribution. The estimates of dispersion and logarithmic fold changes incorporated data-driven prior distributions 63, and the adjusted p-value of the genes was set to <0.05 to detect differentially expressed genes.