Illumina HiSeq 2000 paired end sequencing; Changes in the coding/non-coding transcriptome and DNA methylome that define the Schwann cell repair phenotype after nerve injury
Changes in the coding/non-coding transcriptome and DNA methylome that define the Schwann cell repair phenotype after nerve injury
Sciatic nerves of adult mice were cut or crushed at the sciatic notch. RNA was extracted from samples using Trizol (Invitrogen) according to manufacturer’s instructions with an additional purification step by on-column DNase treatment using the RNase-free DNase Kit (Qiagen) to ensure elimination of any genomic DNA. The integrity and quantity of RNA was determined using Qubit (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent Technologies). 1 µg of total RNA was used to generate RNA-Seq libraries using TruSeq Stranded total RNA sample preparation kit (Illumina) according to the manufacturer’s instructions. Briefly, cytoplasmic and mitochondrial rRNA was removed using biotinylated, target specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, RNA was fragmented using divalent cations under elevated temperature and cleaved fragments were copied onto first strand cDNA using reverse transcriptase and random primers. Next, the second strand was synthesised using DNA Polymersae I and RNase H. cDNA 3’ ends were adenylated and indexed adapters ligated followed by 10 cycles of library amplification. Finally, the libraries were purified using AMPue XP Beads (Beckman Coulter) and quality was evaluated using Agilent 2100 Bioanalyzer. Three libraries were run on a single lane of the HiSeq 2000 platform (Illumina) to generate 100 bp paired-end reads.
Experimental Factor: RNA-seq of coding RNA
protocol
Experimental Factor: cut nerve
injury
Experimental Factor: 7
time