E-MTAB-3707:Sample 13
Illumina HiSeq 2500 paired end sequencing; Gene expression profiling of mouse liver cells
Gene expression profiling of mouse liver cells
Sample 13
14 week old B6CastF1 (C57Bl/6J mother x CAST/Ei father) female mouse were anesthetized and perfused first with PBS, and then with Liver Perfusion Medium (Gibco, Life Technologies). The whole liver was dissected and transferred to 10mL of Liver Digest Medium (Gibco, Life Technologies). The liver was mechanically dissociated, and incubated at 37◦C for 15-20 minutes with continuous shaking (225 rpm). Remaining tissue aggregates were gently triturated until dispersed, and the resulting liver cell suspension centrifuged for five minutes at 1000 rpm (at room temperature). The supernatant was carefully removed and the cells re-suspend in 10 mL of ice-cold Hepatocyte Wash Medium (Gibco, Life Technologies). The centrifugation and re-suspension steps were repeated twice additional times, and in the final re-suspension step, Hepatozyme (Gibco, Life Technologies) media was used. Finally the cells were filtered using a CellTrics 30 μM mesh filter (Partec), and kept on ice until loaded into the C1 IFC unit. All experiments involving mice were approved by the local ethical review committee, and a certificate of designation from the UK Home Office (the national authority for animal experimentation) was obtained. 5000 liver cells were loaded onto a 10-17 μm C1 Single-Cell Auto Prep IFC (Fluidigm), and cell capture was performed according to the manufacturer’s instructions. The capture efficiency was inspected using a microscope, and there were single cells in 70 wells, cell debris in four wells, and more than one cell (or one cell plus debris) in 22 wells. The data from wells that contained more than one cell or debris was subsequently removed from analysis. Upon capture, reverse transcription and cDNA pre-amplification were performed in the 10-17 μm C1 Single-Cell Auto Prep IFC using the SMARTer PCR cDNA Synthesis kit (Clontech) and the Advantage 2 PCR kit (Clontech). 1 μl of the ERCC Spike-In Control Mix (Ambion) in a 1:400 dilution in C1 Loading Reagent was added to the lysis mix for the liver cells. cDNA was harvested, quantified with the Bioanalyzer DNA High-Sensitivity kit (Agilent Technologies), and Nextera libraries prepared using the Nextera XT DNA Sample Preparation Kit and the Nextera Index Kit (Illumina) by following the instructions in the Fluidigm manual (“Using the C1 Single-Cell Auto Prep System to Generate mRNA from Single Cells and Libraries for Sequencing”).
EMBL-EBI
E-MTAB-3707:Sample 13
Experimental Factor: cell 13
individual