Illumina HiSeq 4000 sequencing; A hidden threshold in motor neuron gene networks for survival revealed by modulation of miR-218 dose
A hidden threshold in motor neuron gene networks for survival revealed by modulation of miR-218 dose
Spinal cords were micro-dissected (Leica stereomicroscope) from E12.5 mice carrying a single Hb9::gfp+ allele and were dissociated with papain (papain dissociation kit, Worthington Biochemical) for 30 minutes. Tail clips were simultaneously taken from each embryo for genotyping. Spinal tissue was triturated and centrifuged at 1000rpm for 5 minutes. Dissociated cells were resuspended in 1:1 Neurobasal:DMEM/F12 (without phenol red) with 3% Horse Serum (Invitrogen) and DNase (Worthingon Biochemical) and were passed through a 35µm cell strainer (08-771-23, BD Falcon). Cells were sorted on a Becton Dickinson FACS Vantage SE DiVa using Coherent Sapphire 488nm solid state lasers (200mW). mRNA sequencing libraries were prepared using the TruSeq RNA Library Preparation Kit (v2) according to the manufacturer's instructions (Illumina). Briefly, RNA with polyA+ tails was selected using oligo-dT beads. mRNA was then fragmented and reverse-transcribed into cDNA. cDNA was end-repaired, index adapter-ligated and PCR amplified. AMPure XP beads (Beckman Coulter) were used to purify nucleic acids after each step.  Stranded mRNA-Seq libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's instructions (Illumina). Briefly, poly-A RNA was isolated using magnetic beads conjugated to poly-T oligos. mRNA was then fragmented and reverse-transcribed into cDNA. dUTPs were incorporated, followed by second strand cDNA synthesis. dUTP-incorporated second strand was not amplified. cDNA was then end-repaired, index adapter-ligated and PCR amplified. AMPure XP beads (Beckman Coulter) were used to purify nucleic acid after each steps of the library prep.
Experimental Factor: genotype
Hb9::gfp;miR-218-2KO/KO
Experimental Factor: protocol
bulk RNAseq