SRX2900170 GSM2656712 SRP108903 SRS2267415 GSM2656712 OTHER GENOMIC other The first step of insertion mapping involves using a single biotinylated primer that binds in the known sequence of the inserted vector. The primer will extend, amplify and generate many molecules of the biotinylated ssDNA including the vector sequence and its point of integration into the genome. This ssDNA PCR was performed on genomic DNA from parental HT1080 cells, bulk cultures obtained from co-transfections of seamless vector attL4X-PGKssPuro with pCMVssIna and bulk cultures obtained from co-transfections of seamless vector attL4X-PGKssPuro with pCMVssInt-C3CNLS. Biotinylated primer Puro fwd 127 was used to generate the ssDNA extending from puro cassette to the junction of insertion of vector into the human genome. PCR was performed using Bioline Taq polymerase with Tris-based buffer (Vivantis) and the reaction conditions includes 95◦C, 120 seconds; 95◦C, 5 seconds; 50◦C, 30 seconds, 72◦C, 120 seconds; 50 cycles. The second step includes the capture of ssDNA onto the streptavidin dynabeads (M-280 Streptavidin, Thermo Fisher Scientific), which includes the washing of dynabeads with 1X PCR buffer (Tris-based) followed by incubation of the PCR product generated in the first step with dynabeads for 3 hours on roller at room temperature. The phosphorylated primer pETF2-PH was used as an adaptor and 5’ adenylation was done using kit (NEB E2610S). The ligation of the 5’ adenylated oligo with captured ssDNA was done with the help of 5’ AppDNA ligase (NEB) at 65◦C for an hour. Semi-nested PCR was performed on beads with forward primer Puro fwd 128 and reverse primer petF2RC using Taq Polymerase (Bioline) and non-Tris based buffer (Bioline). PCR reaction conditions includes 95◦C, 120 seconds; 95◦C, 5 seconds; 55◦C, 30 seconds, 72◦C, 120 seconds; 34 cycles. Further, a nested PCR was carried with forward primer bpa1 and cs_attH4X_R1 and the PCR products were analyzed on 1% agarose in 0.5% TBE buffer. The PCR amplicon with the expected size was excised and purified. The amplicon was sheared and ligated with standard illumina adaptors for shortgun sequencing with 100 Paired End reads. Illumina MiSeq gds 302656712 GSM2656712 GEO Accession GSM2656712