MEF5 whole exome seq
two groups of mouse embryonic fibroblasts (MEFs) from different parents were treated with 500 ug/ml ethylnitrourea (ENU) for 30 min, and further cultured in DMEM for 72 hours before being harvested for single cell isolation and sequencing assay. For all our single cell RNA-seq data (MEF1-10): basically we performed RT-PCR following a published protocol (Hartmann & Kline, 2006, NAR, 34:e143) with few modifications. Instead of polyC PCR primer, we incorporated a MmeI (a IIS restriction enzyme, which cleaves 20bp downstream of the recognition site) site in the homopolymer PCR primer, for the purpose to trim polyC sequence after amplification. After quality validation by BioAnalyzer assay, the global mRNA amplicons were randomly fragmented, and subjected to multiplexed, paired-end tagged library construction according to Meyer & Kircher (2010) Cold Spring Harb Protoc prot 5448. For MEF bulk, whole RNAs were extracted using a Qiagen RNeasy spin-column. After RT using random primers, the second strand syntheses were performed using a New England Biolab kit. The resultant double strand cDNA were also subjected to random fragmentation and PET library construction.  All those samples were subsequently subjected to an Illumina 2500 Rapid PE run, with a readout length of 150bp.   