title: NextSeq 550 paired end sequencing; RNA-Seq of Diversity Outbred mouse embryonic stem cells
design: RNA-Seq of Diversity Outbred mouse embryonic stem cells
library_construction_protocol: ESCs were derived from random blastocysts of Diversity Outbred (Jackson Laboratory #009376) mice using previously published protocols (Czechanski et al. 2014 Nature Protocols). As the ES cells expanded, they were transferred into 24 well plates, followed by 6 well plates, and finally 10cM dishes, all without feeder cells but in the same media.  Cells were maintained in ES medium (ESM, 1i+LIF: 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, approximately 2000U/ml LIF, and 3uM CHIR99021). For each mESC line, one 15cm dish of cells was grown to near confluence, washed 3x with PBS, and mechanically harvested to produce 80-150mg wet weight cell pellets (~50M cells). About 100k cells from each frozen cell pellet were used for RNA sequencing.    Cell lines were untreated. Total RNA was extracted using the Quick-RNA 96 well format kit (Zymo Research) with in-column DNase treatment.  Sequencing libraries were prepared using the TruSeq Stranded mRNA HT kit (Illumina, Cat no. 20020595) and included ribosomal RNA reduction and poly-A selection, enzymatic fragmentation, cDNA synthesis from random hexamer priming, adapter ligation and PCR amplification steps to generate indexed, stranded mRNA-seq libraries. Libraries were checked for quality and quantitated with the Agilent Bioanalyzer, and samples that failed QC were repeated starting from the cryovial stage.