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Fig. S2 Identification and characterization of lincRNAs involved in mESC self-renewal. (A) Oct4-GFP mESCs were transduced with the shRNA library and selected for 4 days in ESC medium. The GFP+ (undifferentiated) and GFP- (differentiated) cells were isolated by FACS and DNA was extracted. (B) The scheme to identify the candidate lincRNAs essential for self-renewal. A total of 3265 shRNAs were represented in the three GFP- biological replicates and 3115 in the GFP+ replicates. For each shRNA in the common set of 2788, the ratio of {Average[GFP-]/Average[GFP+]} was calculated. The ratio for the nontargeting control shRNA was 1.29. Candidate lincRNA genes were selected if at least two shRNAs were present at a GFP-/GFP+ ratio >2.5 or if one shRNA was present at a ratio >3. (C) AP staining of mESCs on day 4 after knockdown of 21 selected lincRNA genes. Control cells were transduced with a nontargeting shRNA (shCtrl) or an Oct4-specific shRNA. Two shRNAs were tested for each lincRNA. Scale bars, 100 µm.