Sample Detail

TitlePB366_07
DescriptionProtocols: 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: Dulbecco'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.

Organism Info

Taxon ID10090
Common Namehouse mouse
Scientific NameMus musculus
Anonymized Name
Individual Name