title: HEK293 PGBD5 + PB-EF1-NEO - MiSeq
design: Flanking sequence exponential anchored (FLEA) PCR  To amplify genomic transposon integration sites, we modified flanking sequence exponential anchored (FLEA) PCR (5), as described in Supp. Fig. S8. First, linear extension PCR was performed using 2 μg of genomic DNA and 100 nM biotinylated linear primer using the Platinum HiFidelity PCR mix, according to manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA, USA). Linear extension parameters for PCR were: 95 ˚C (45 sec), 62 ˚C (45 sec), 72 ˚C (3 min) for 30 cycles. Reaction products were purified by diluting the samples in a total volume of 200 μl of nuclease-free water and centrifugation using the Amicon Ultra 0.5 ml 100K at 12,000 g for 10 minutes at room temperature (EMD Millipore, Billerica, MA, USA)  purification. Retentate was bound to streptavidin ferromagnetic beads on a shaker at room temperature overnight (Dynal, Oslo, Norway). Beads were washed with 40 μ l of washing buffer (Kilobase binder kit; Dynal), then water, then 0.1 N NaOH and finally with water again.   To anneal the anchor primer, washed beads were resuspended in a total volume of 20 μl containing 5 μM anchor primer, 500 nM dNTP, and T7 DNA polymerase buffer (New England Biolabs, Beverly, MA, USA). Samples were placed in a heating block pre-heated to 85 ˚C, and allowed to passively cool to 37 ˚C. Once annealed, 10 units of T7 DNA polymerase (New England Biolabs) was added and the mixtures were incubated for 1 h at 37 °C. Next, the beads washed 5 times in water.   To exponentially amplify the purified products, beads were resuspended in a total volume of 50 μl containing 500 nM of exponential and Transposon1 primers, and the Platinum HiFidelity PCR mix. PCR was performed with the following parameters: 95 °C for 5 min, followed by 35 cycles of 95 °C for 45 sec, 62 °C for 45 sec and 72 °C for 3 min. PCR products were purified using the Invitrogen PCR purification kit (Invitrogen Corp., Carlsbad, CA, USA). Second nested PCR was performed using 1/50th of the first exponential PCR product as template using the Platinum HiFidelity PCR with 500 nM of exponential and Transposon2 primers. PCR was performed with the following parameters: 35 cycles of 95 °C for 45 sec, 62 °C for 45 sec and 72 °C for 3 min. Final PCR products were purified using the Invitrogen PCR purification kit, according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA, USA).    Sequencing of transposon reporter integration sites  Equimolar amounts of purified amplicons were pooled according to concentration provided by qubit measurement (Invitrogen Carlsbad, CA) and sized on a 2100 BioAanalyzer Instrument (Agilent Technologies,  Santa Clara, CA).  The sequencing library construction was performed using the KAPA Hyper pre Kits Catalog (#KK8504, KAPA biosystems, Wilmington, MA) and 12 indexed Illumina adaptors from IDT (Coralville, IO), strictly following manufacturer’s instructions.  After quantification and sizing, libraries were pooled for sequencing on a miseq (pooled library input at 10pM) on a 300/300 paired end run.