Illumina HiSeq 4000 paired end 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). Sorted cells were spun down at 300 rcf for 2 minutes, and 370uL were removed to leave remaining 130 uL. The cells were resuspended in this remaining media. 32.5uL of the resuspended cells were loaded into 10X Chromium Controller using Chromium Single Cell 3' v2 reagents. Sequencing library was prepared following the manufacturer's instructions (10X Genomics).
Experimental Factor: genotype
Hb9:gfp
Experimental Factor: protocol
single cell RNAseq