Embryonic Stem Cell Transcriptome Analysis

Independent biological replicates of WT (n = 2), calr^−/− (n = 5), and calr truncation mutants NP (n = 3) and PC (n = 3) were cultured on 0.1 % gelatin-coated 10 cm dishes in 7.5 % FBS in DMEM supplemented with L-glutamine, NEAA, penicillin/streptomycin, LIF, and BME. Media was changed regularly every 24 or 48 h. Cells reaching ~80 % confluency were passaged 1:10 to maintain proliferative undifferentiated state of all cell lines, as previously performed [18 (link)]. Total RNA isolation from embryonic stem cells was performed using the Micro-to-Midi Total RNA Purification System (Invitrogen, Carlsbad, CA) for analysis of transcriptome dynamics. Double stranded complementary cDNA and labeled complementary cRNA were obtained from isolated total RNA, with the latter hybridized against the Mouse 430 2.0 GeneChip (Affymetrix). Arrays were scanned using an argon-ion laser, and visualized using MAS 5.0 Affymetrix software to assess quality of hybridization. Data were deposited to the Gene Expression Omnibus (GEO) repository under accession number GSE13805, with relevant updates.