DNeasy Kit from Qiagen Custom; shRNA amplicon After recovery, 5x10e6 cells were harvested and gDNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The shRNA cassettes were isolated from genomic DNA with a first PCR reaction using the following primers: pLKO ForHTm primer (GGGCCTATTTCCCATGATTCCTTC), and Rev-pLKO.1 (CCAAAGTGGATCTCTGCTGTCCC). After verification of a single PCR product at 497 bp size, PCR reactions were pooled and cleaned up with QIAquick PCR Purification Kit (Qiagen). Next, 1microg of the resulting DNA was digested with XhoI overnight at 37C (for ligation of barcode linkers to allow multiplex sequencing), gel excised and the appropriate band was isolated using QIAquick Gel Extraction Kit (Qiagen). Selected barcode linkers were added to ligation reactions with 100 ng of the purified XhoI fragment. Ligations were performed overnight at 16C and run on 2% TAE agarose gel. The resulting ligation product was excised from the gel and purified as before. A second PCR was performed for addition of Illumina adapters with PCR2 primers (PCR2 Fwd: CAAGCAGAAGACGGCATACGATGGAAAGGACGAAACACCGG, PCR2 rev: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT). These PCR reactions were run again on a 2% TAE agarose gel, and the appropriate final product was excised and purified as above. Before sequencing, we assessed the purity and quantity of our sequencing libraries with the Bioanalyzer High Sensitivity DNA Kit (Agilent), and confirmed the presence of only one DNA fragment.
GEO Accession
GSM2298386