GSM2195659: Ly49G+ve/Ly49I-ve NK Rep 2; Mus musculus; RNA-Seq
Single cell suspensions were prepared from the spleens of 20 C57Bl/6Ncr mice.  NK cells were enriched using the MACS NK Cell purification system (Miltenyi Biotec).  The Fc receptors of the resulting populations of enriched NK cells were blocked with 2.4G2 and the cells were stained with antibodies to CD3, NK1.1 (eBioscience), Ly49G2 (4D11), and Ly49C/I (5E6).  CD3-, NK1.1+,Ly49G+, Ly49C/I- and CD3-, NK1.1+,Ly49G-, Ly49C/I+ cells were sorted on a FACS Aria (BD Biosciences). Stranded RNA-seq libraries were constructed from 0.4 µg total RNA using the TruSeq Stranded Total RNA Sample Prep Kits (Illumina). Equal volumes of individual libraries were pooled and run on a MiSeq benchtop DNA sequencer (Illumina).  The libraries were then repooled to equimolar concentrations based on the MiSeq demultiplexing results. The final pooled library was sequenced on a HiSeq2000 sequencer (Illumina) using TruSeq Version 3 chemistry. A minimum of 40 million 100 base read pairs were generated for each library.