strain that es cell-line is derived from: 129S4/SvJae original mes cell line: J1 cell-line: SclKO mESC cell type: ES cell derived day4 EB (embryoid body) Flk1+ mesodermal cells treatment/cell subtype: EB formation, FACS sorting for Flk1+ mesodermal cells
Treatment protocol
ES cells were differentiated into Flk1+ cells by 2 days of culture in pre-EB media (IMDM with the addition of L-glutamine, 1% of penicillin and streptomycine, 1.5 x 10-4 M monothiolglycerol (MTG), 15% of FBS and 10ng/ml LIF; followed by plateing 100 000 cells per well to low attachment 6-well plates (Costar) in EB media ((IMDM with the addition of 15% of FBS, L-glutamine, 1% of penicillin and streptomycine, 1.5 x 10-4 M MTG, 3mg/ml human transferrin (Roche) and 2.5mg/ml ascorbic acid (Sigma) and clulturing the cells for 4 days. Cells were digested enzymatically and FACS sorted for desired populations.
Growth protocol
Standard ES cell culture media with DMEM (Cellgro), 15% serum (Hyclone or Omega) and 10ng/ml LIF (Millipore) and gelatin coated dishes were used to maintain SclhCD4 knock-in (Chung et al., 2002), SclKO (Porcher et al., 1996) and WT ES cells.
Extracted molecule
total RNA
Extraction protocol
Day 4 EB cells were sorted on a BD FACS Aria II (BD Biosciences) using Flk1-PE and hCD4 (clone S3.5, Invitrogen) antibodies to collect WT Flk1+hCD4+(Scl+), WT Flk1+hCD4-(Scl-) and SclKO Flk1+ cell polulations.
Label
biotin
Label protocol
Affymetrix standard labeling kit
Hybridization protocol
Affymetrix standard hybridization protocol
Scan protocol
Affymetrix standard scan protocol
Data processing
The R package Limma (Gentleman, 2005) provided through the open source project Bioconductor (Gentleman et al., 2004) was used for assessing differential expression. To calculate absolute mRNA expression levels, the RMA (Robust Multiarray Averaging (Bolstad et al., 2003)) method (provided through R library affy) was used to obtain background adjusted, quantile normalized and probe level data summarized values for all probe sets. The Affymetrix Mouse Genome 430 2.0 Array GeneChip platform was used for the analysis. Official gene symbols for probe sets were obtained using the Bioconductor annotation database mouse4302.db. The mas5calls algorithm (Liu et al., 2006) through the R package affy was used for calculating PMA detection calls for each array sample. The algorithm employs a signed rank test to consider the significance of the difference between the PM and MM values for each probeset and returns a detection p-value that is used to flag a transcript as 'Present', 'Marginal' or 'Absent' (P/M/A). Probes that were 'Absent' for all samples analyzes were excluded from further analysis. Differentially expressed genes were uploaded into the DAVID (Huang et al., 2007) interface to identify significantly over-represented functional GO biological process categories.
