Mouse 430 2.0 genechip

Independently derived 70Z/3 lines infected with MSCV, or MSCV-miR-24-2 were generated previously[65 (link)]. Four MSCV lines and 2 MSCV-miR-24-2 lines were examined. Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA). RNA quality was evaluated using the Lab-on-a-Chip Bioanalyzer 2100 (Agilent, Palo Alto, CA). Biotinylated cRNA was prepared according to the standard Affymetrix protocol using the Superscript Choice System (Invitrogen) and the RNA transcript labeling kit (ENZO, Farmingdale, NY) for cRNA preparation. Following fragmentation 10ug of cRNA was hybridized to Affymetrix Mouse 430 2.0 Gene Chip. Microarray analysis was performed using the Affymetrix GeneArray Scanner G2500A. The Affymetrix Expression Console Software Version 1.4.0 was used to create summarized expression values (CHP-files) from expression array feature intensities (CEL-files). Raw data were normalized with robust multichip analysis (RMA) with Affymetrix transcriptome analysis console (TAC) software. DNA microarray and sample annotation data were deposited in GEO under the accession number GSE65874.

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.

A microarray dataset of mouse auditory nerve development from our group (NCBI Gene Expression Omnibus accession GSE59417; Lang et al., 2015 (link)) was used for comparative analysis. The dataset contains expression data for auditory nerve samples collected at P0, 3, 7, 10, 14 and 21 analyzed by Mouse 430 2.0 GeneChip (Affymetrix, Santa Clara, CA, USA). Raw hybridization data was normalized independently by both Robust Multi-array Average and MicroArray Suite 5.0 algorithms using Expression Console Software (Affymetrix). Differential expression was defined as absolute signal log ratio >0.5, ≥50% present gene detection scores and p ≤ 0.05 (Student’s t-test, unpaired) for P7 vs. P0 (identifying 2391 probe sets). False discovery rate was estimated at 1.7% based on iterative comparisons involving randomized sample group assignments. Among the 2391 probe sets, 1878 analysis-ready genes were found. Functional analysis and canonical function identification was assessed with Ingenuity Pathway Analysis Software version (IPA^®; QIAGEN, Redwood City, CA, USA).