Illumina HiSeq 2000 paired end sequencing; Single cell mRNA -sequencing reveals cell-to-cell variation in three mouse ES cell culture conditions
Single cell mRNA -sequencing reveals cell-to-cell variation in three mouse ES cell culture conditions
Cells were grown in one of 3 different media: Serum-containing media: Knockout DMEM (Gibco), 1X penicillin-streptomycin-glutamine (Gibco), 1X non-essential amino acids (Gibco), 100 U/ml recombinant human leukemia inhibitory factor (Millipore), 15% fetal bovine serum (HyClone), 0.1mM _-mercaptoethanol (Sigma). Standard 2i media: N2B27 basal media (NDiff 227, StemCells), 100 U/ml recombinant human leukemia inhibitory factor (Millipore), 1 _M PD0325901 (Stemgent), 3 _M CHIR99021 (Stemgent). Alternative 2i media: N2B27 basal media (NDiff 227, StemCells), 100 U/ml recombinant human leukemia inhibitory factor (Millipore), 1 _M CGP77675 (Sigma), 3 _M CHIR99021 (Stemgent). (SMARTer) reverse transcription and cDNA preamplification were performed in the 10-17 microns Fluidigm C1 Single-Cell Auto Prep IFC using the SMARTer PCR cDNA Synthesis kit (Clontech) and the Advantage 2 PCR kit (RNAEasy) RNA was extracted usin RNAEasy kit, Qiagen (Nextera XT) For each culture condition, 4000 cells were loaded onto a 10-17 micron Fluidigm C1 Single-Cell Auto Prep IFC and cell capture was performed according to the manufacturer's instructions. Nextera libraries were according to “Using the C1TM Single-Cell Auto Prep System to Generate mRNA from Single Cells and Libraries for Sequencing”. (TruSeq) Bulk mRNA sequencing libraries were prepared and sequenced at the Wellcome Trust Sanger Institute sample preparation pipeline using Illumina's TruSeq RNA Sample Preparation v2 kit. RNA was extracted from 1-2mln cells using Qiagen RNA Purification Kit on QiaCube robot. Quality of RNA sample was checked using gel electrophoresis. For library preparation poly-A RNA was purified from total RNA using oligo-dT magnetic pull-down, subsequently mRNA was fragmented using metal-ion catalyzed hydrolysis. cDNA was synthesized using random priming, end repair was performed to obtain blunt ends and A-tailing was done to enable subsequent ligation of Illumina paired-end sequencing adapters. Samples were multiplexed at this stage. Resulting library was amplified using 10 cycles of PCR using Kapa Hifi polymerase instead of polymerase provided by Illumina in TruSeq kit
Experimental Factor: growth condition
standard 2i media + LIF