GSM5965305: 7118 HA; Mus musculus; RNA-Seq
Astrocyte ribosome-associated mRNA isolation: Fourteen days after complete crush SCI or 24 hours following IP injection of LPS, spinal cords of wild-type, Stat3-astro-cKO or Smarca4-astro-cKO mice were perfused with ice cold saline and spinal cords rapidly dissected and frozen on dry ice. For SCI samples, the central 3 mm of the lower thoracic lesion including the lesion core and 1 mm rostral and caudal were then rapidly removed and snap frozen in liquid nitrogen. Anatomically equivalent regions of spinal cord were taken from mice treated with LPS or from genotype-matched healthy controls. Frozen tissue samples were processed either for RiboTag analysis via haemagglutinin (HA) immunoprecipitation and purification of astrocyte ribosome-associated mRNA using standard methods. Astrocyte RNA Integrity was analysed by a 2100 Bioanalyzer (Agilent) using the RNA Pico chip, RIN ≥ 8.9 for all samples. cDNA was generated from 10 ng of RNA using the Universal plus mRNA-Seq Kit (Nugen). Multiplexed sequencing was performed with a NovaSeq 6000 sequencer (Illumina) on a NovaSeq S2 flow cell to produce 50 base-pair paired-end reads.