title: Illumina HiSeq 2500 sequencing; Genome-wide  profiling of Alternative polyadenylation sites in mouse female germline stem cells
design: Genome-wide  profiling of Alternative polyadenylation sites in mouse female germline stem cells
library_construction_protocol: Mouse FGSCs were maintained on 1% gelatin-coated dishes in minimum essential medium  medium (MEM-), supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 1 mM sodium pyruvate, 1 mM non-essential amino acids, 1 mM l-glutamine, 0.1 mM -mercaptoethanol (Sigma), 20 ng/ml LIF (Leukemia Inhibitory Factor, Peprotech), 10 ng/ml EGF (mouse epidermal growth factor; Peprotech), 10 ng/ml human GDNF (glial cell line-derived neurotrophic factor; Peprotech), 10 ng/ml human bFGF (basic fibroblast growth factor; Peprotech). The FGSC medium is steriled by filtration using 0.22m syring-driven filter unit (Millipore).And the medium was changed every 2 days and cells were subcultured using trypsin every 3 days at a 1:23 dilution. All cultures were maintained at 37 C in a 5% CO2 atmosphere. Mouse feeder free 129S4/SvJae ES cells were maintained on gelatin-coated dishes in Glasgow Minimum Essential medium (GMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum, 0.1mM -mercaptoethanol (GIBCO), 2 mM l-glutamine, 0.1 mM MEM non-essential amino acid, 5,000 units ml1 penicillin/streptomycin and 1,000 units ml1 of ESGRO (Chemicon) under feeder-free conditions. Total RNA of FGSC or ESC was extracted using TRIzol regent (Invitrogen) and resuspend in RNase free water. And 1ug total RNA was used to perform reverse transcription with Oligo-dT and random primer using SuperScript III reverse transcriptase(Invitrogen) Total RNA was extract with TRIzol Reagent . And 50 g total RNA was incubated with streptavidin dynabeads coated with biotinylated GsuI-oligo (dT) primer. The first strand cDNA was synthesized with SuperScript III (Invitrogen) and dNTP mix containing 5-methylated-dCTP instead of dCTP. Then the resulting DNA/RNA hybrids were treated with RNase H and DNA polymerase I before dsDNA was generated. Next, the resulting dsDNA was fragmentated to 200500 bp with fragmentase (NEB). 3terminal fragments were released from beads through Gsu I digestion, which were subjected to deep sequencing by Illumina HiSeq2000.
attributes.tag: Experimental Factor: female germ line stem cell
attributes.value: cell type
attributes.tag: Experimental Factor: female germline stem cell line
attributes.value: cell line