title: GSM4929945: ARC99; Mus musculus; RNA-Seq
library_construction_protocol: Mice were transcardially perfused with phosphate-buffered saline (PBS). The entire spinal cord was rapidly isolated by hydraulic extrusion and then the lumbar segment was further dissected for subsequent analysis (Injury L3-L6, ALS entire lumbar cord). Spinal cord samples were snap frozen in liquid nitrogen. Tissues were placed into IP buffer (50 mM Tris, pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40 [NP-40] supplemented with 200 U/mL Promega RNasin, 1 mg/mL heparin, 100µg/mL cycloheximide, and protease inhibitor mixture [Sigma-Aldrich]) and subjected to Dounce homogenization. Samples were then rotated at 4°C for 15-20 minutes followed by centrifugation at 10,000 x g for 10 min at 4°C to generate a postmitochondrial supernatant. A 50µl aliquot of the input was saved and processed separately for RNA isolation. The remainder of the sample was precleared with Protein G magnetic beads (25 µl of beads per 200 µl of supernatant, New England Biolabs) for 1 hour under constant rotation at 4°C. After preclearing, samples were separated from the magnetic beads and incubated with an HA antibody (1:150 dilution, #MMS-101P, BioLegend) by toppling at 4°C for 4 hours. Protein G magnetic beads were then added and allowed to incubate under rotation at 4°C overnight. The IP product associated with the magnetic beads was then washed 3 times in high salt buffer (50 mM Tris, pH 7.4, 300 mM KCl, 12 mM MgCl2, 1% NP-40 supplemented with 100 µg/mL cycloheximide). RNA associated with the HA IP and from the input lysates was isolated using the RNeasy Plus Micro Kit (Qiagen) per manufacturer instructions. The purity and quality of isolated RNA was confirmed by Bioanalyzer or TapeStation (Agilent Genomics). SMART-Seq v4 Ultra Low input RNA kit (Takara) was used to synthesize cDNA from 4ng of RNA using 11 cycles of cDNA amplification, according to manufacturer's protocols. The cDNA was sheared by Covaris and quantitated by Qbit dsDNA HS Assay kit (ThermoFisher). Libraries were prepared from 5 ng cDNA using SMARTer ThruPLEX DNA-Seq kit (Takara) with 8 cycles of PCR according to manufacturer's protocols. Final libraries were checked for quality and quantity by the Bioanalyzer or TapeStation (Agilent Genomics) and qPCR using Kapa's library quantification kit for Illumina Sequencing platforms (#KK4835, Kapa Biosystems).
attributes.tag: GEO Accession
attributes.value: GSM4929945