mapping reads (inverse PCR) of MED30 TRIP pool B, replicate 2
Protocol by Akthar et al. 2014 (Nature protocols) was used for inverse PCR to locate sites of barcoded reporter integrations. 2 g of gDNA was digested with 20 units of DpnII (New England Biolabs) overnight at 37C in a volume of 100 l. Subsequently, 600 ng of purified digested DNA was self-ligated with 40 units of high concentration T4 DNA ligase (Promega) overnight at 4C in a volume of 400 l (2 times for each technical replicate of the TRIP pool). The ligation reactions were phenol/chloroform/isoamylalcohol extracted and ethanol precipitated. DNA pellets were dissolved in 30 l of water. 5 l of each sample was used as a template for amplification of fragments containing both the barcodes and flanking genomic DNA regions (two PCR replicates were performed each with a different index). PCR was performed in three rounds and purified products were directly used for high throughput Illumina paired-end sequencing.