GSM1534005: MRG_minus_rep2_shRNA screen; Homo sapiens; OTHER
shRNA screen: We selected 244 transcription factors and epigenetic modifiers that were differentially or continuously highly expressed during our in vitro differentiation time course in an otherwise unbiased fashion (Extended Data Table 4). In addition, we included GFP, RFP, lacZ and luciferase as internal controls.  We then obtained a sub-pool of the human 45K shRNA pool6 distributed by the Broad Institute Genomic Perturbations Platform and the RNAi Consortium (TRC) against these genes. For each gene, 5 distinct shRNAs were included as well as 5 scrambled and 3 empty control vectors, amounting to a total of 1230+8 shRNAs. The plasmid for shRNA expression under the control of the constitutive U6 shRNA promoter was the lentiviral vector pLKO.1. shRNA pool production and infection conditions were performed as previously described6. Subsequently, we performed calibration experiments to determine to optimal combination of MOI and Puromyocin concentration to ensure efficient selection. We identified MOI 0.4 and 1ug/ml of Puromycin as optimal parameters for all stages. We then infected 26 million cells at each stage of NE, ERG and MRG to ensure sufficient shRNA integration events to recover the complexity of the shRNA library. 24h post infection and prior to full expression but after integration of the lentivirus into the genome we collected 3 million cells to determine our baseline shRNA library representation. Subsequently, we subjected the cells to 5 days of Puromycin selection and then FACS sorted the resulting populations into HES5+ and HES5- compartments. We assessed the representation of the shRNA library in each of the 9 populations by retrieving all shRNA integration events from genomic DNA isolated from each sample using PCR followed by next generation sequencing as previously described (Strezoska, Z. et al 2012). More specifically, we performed two rounds of PCR using the following primers for the primary PCR: Primary R :CTTTAGTTTGTATGTCTGTTGCTATTAT; Primary F: AATGGACTATCATATGCTTACCGTAAC.  For the second, nested PCR we used: Nested F: GGCTTTATATATCTTGTGGAAAGGA; Nested R: GGATGAATACTGCCATTTGTCTC.
GEO Accession
GSM1534005