title: Illumina HiSeq 2000 paired end sequencing; RNA-Seq of mouse embryonic stem cells derived from genetically diverse inbred mouse strains
design: RNA-Seq of mouse embryonic stem cells derived from genetically diverse inbred mouse strains
library_construction_protocol: Euploid (>70%), germline competent, male mESCs were derived from blastocysts of genetically diverse inbred mice.  Cells were maintained in 2i+LIF media in the presence of mitotically inactivated mouse embryonic fibroblasts (MEFs, C57BL/6J). 2i+LIF media contained Dulbeccoâ€™s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum, 100 U/mL Penicillin-Streptomycin, 2mM GlutaMAX, 0.1mM non-essential amino acids, 1mM sodium pyruvate, 0.1mM 2-mercaptoethanol, 500pM LIF, 1uM PD0325901, and 3uM CHIR99021. Prior to harvesting cells for RNA collection, MEFs were removed through sequential plating (2X, 1 hour) onto gelatin-coated dishes. Cell lines were untreated. RNA was harvested using the RNeasy (Qiagen) RNA extraction kit.  Poly(A) RNA-seq libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina), including the addition of unique barcode sequences.