title: GSM5833422: Host Spinal cord astrocytes from 73.12 Gffap-Ribotag rep 9; Mus musculus; RNA-Seq
library_construction_protocol: SCI tissue or cell pellet samples were homogenized in supplemented homogenization buffer (50 mM Tris pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM Dithiothreitol (DTT), 1X Protease inhibitors, 200 U/ml RNAsin, 100 mg/ml Cyclohexamide, 1 mg/ml Heparin) using 2mL glass Dounce tissue grinders (D8938-1SET, Sigma). Tissue homogenates were centrifuged to remove tissue debris before being incubated with Anti-HA.11 Epitope Tag Antibody (Biolegend, Cat# 901515) for 4 hours in a microcentrifuge tube on a microtube rotator kept at 4°C. At the end of 4 hours, IP solutions were combined with Pierce A/G Magnetic Beads (Thermofisher, #PI88803) and incubated overnight on a microtube rotator at 4°C. On the second day, magnetic beads were washed three times with high salt solution (50 mM Tris pH 7.4, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM Dithiothreitol (DTT), 100 mg/ml Cyclohexamide). Unpurified RNA was collected from the magnetic beads by addition of RLT Plus buffer with BME and vigorous vortexing. RNA was then purified using RNeasy Plus Mini (for in vitro cell pellets) or Micro Kits (for spinal cord tissue) (QIAGEN Cat# 74134 and 74034). Total mRNA derived from the RiboTag IP was quantified using an automated electrophoresis bioanalyzer. RNA samples with RNA integrity numbers (RIN) greater than seven were processed for RNA-Sequencing. Sequencing was performed on poly-A selected libraries using Illumina NovaSeq S2 (housed in the UCLA Technology Center for Genomics & Bioinformatics) using pair end reads (2x50 – 50bp length) with an average of 50-100M reads per sample. The workflow for library construciton involved poly(A) RNA selection, RNA fragmentation and double-stranded cDNA synthesis using a mixture of random and oligo(dT) priming, followed by end repair to generate blunt ends, adaptor ligation, strand selection, and PCR amplification to produce the final library.