Illumina HiSeq 4000 paired end sequencing; Lineage-dependent spatial and functional organisation of the mammalian enteric nervous system
Lineage-dependent spatial and functional organisation of the mammalian enteric nervous system
tdT+ cells were separated using the FACSAria Fusion (BD Biosciences) cell sorter with the 100µm nozzle at 26psi and collected in 2% FBS in OptiMEM (Life Technologies). Dead cells were excluded with a cell viability dye (Zombie Aqua, BioLegend) that was added to the single cell suspension (2 ng/ml). The Fluidigm C1 automated microfluidic system was used to randomly capture 192 individual cells into 5-10µm chips. SER93|tdT is a transgenic line Sox10-CreERT2;Rosa26-Tomato (R26-CAG-Ai14). Information regarding its generation can be found in Laranjeira et al., 2011. SER93|tdT embryos were exposed to Tamoxifen at E12.0 in utero (100µg/gr of dam body weight) and harvested 24 hours later. Individual small intestines were digested with Dispase/Collagenase (1 mg/ml, Roche, 296638) at 37°C for 15 minutes and manually triturated to obtain a single-cell suspension. The SMARTer Ultralow RNA kit (Clontech #634833) was used to reverse transcribe poly (A) RNA and amplify cDNA. ERCC spike-ins (Life Technologies #4456740) were added at a dilution of 1:80000. cDNA concentration of each cell sample was quantified using the Promega Quantifluor dsDNA on Glomax system and quality of random samples was checked on high-sensitivity DNA chip on Agilent Bioanalyser 2100. Amplified cDNA (0.125-0.375 ng) was used for sequencing libraries with the Illumina Nextera XT DNA kit (Illumina #FC-131-1096).
Experimental Factor: GA60_D03_27
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