title: GSM4197359: E14 cells, TC_LA_no_ISceI_rep3; Mus musculus; OTHER
library_construction_protocol: Genomic DNA from transfected cells was extracted using DNeasy Blood and Tissue Kit (Qiagen, #69504) Inverse PCR: 10 μg genomic DNA from transfected mESCs were digested using 10 μl 10 U/μL NlaIII (NEB, #R0125S) in a 50 μL final volume for 3 hours at 37­ °C. The reaction was heat-inactivated at 65­ °C for 20 minutes. The reaction was diluted to a final volume of 1.8 mL in 1X T4 ligase buffer (Thermo Fisher Scientific, #EL0013) to favor self-ligation events, and ligation was carried out with 600 U of T4 ligase (Thermo Fisher Scientific, #EL0013) at 16­°C overnight. After ligation, samples were precipitated by ethanol, pellets were resuspended in water and column-purified (QIAGEN, QIAquick PCR purification kit #28104) eluting with 100 uL EB.  Non circularized templates were eliminated by 2 h digestion at 37­°C with Plasmid-safe DNAse (Epicentre, #E3101K), the enzyme was inactivated by heating for 30 minutes at 70°C. The product was column-purified (QIAGEN, QIAquick PCR purification kit #28104). The backbone of the TRIP reporters contains a I-CeuI site outside the transposable cassette, taking advantage of this, non integrated plasmids were cut by 2 h digestion at 37­ °C with I-CeuI restriction enzyme (NEB, R0699S) in a total volume of 70 ul  followed by 20 minutes heat inactivation at 65­ °C. All enzymatic reactions were carried out in the recommended manufacturer's buffer. For the first round of nested PCR, 10μL of I-CeuI-digestion products was mixed in 50 μL standard Phusion polymerase reaction mix (Thermo Fisher Scientific, F530S) in GC buffer, with 0.1 μM primers IR1,2 (annealing to IR/DR sequence). The cycling conditions were as follows: 98 °C for 5 min; 98°C for 20 s, 60°C for 1 min and 72°C for 5 min (1 cycle); 98°C for 20 s, 60°C for 1 min and 72°C for 2 min (20 cycles); and 72°C for 5 min. Products of the reaction were purified using Agencourt AMPure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water . For the second round of nested PCR, 37 μL of the products was diluted to 50 μL of standard Phusion polymerase reaction mix in GC buffer with 0.1 μM primer M1 (annealing to the Illumina PE1.0 primer) and one of indexing primers M2,3. The cycling conditions were as follows: 98°C for 2 min; 98°C for 20 s, 60°C for 1 min and 72°C for 1 min (10 cycles); and 72°C for 5 min. Primers M2,3 added the Illumina PE2.0 primer and one of indices to the amplicons. Products of the reaction were purified using Agencourt AMPure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. PCR products ran as a smear on agarose gel. The smears were specific, because they failed to appear when the mESCs were not transfected. To eliminate I-SceI sites that were not cut during DSB induction and limit the size of PCR extension, 2 μg genomic DNA from mESCs (both, with DSB induction and control without the induction) were digested using 1 μl 100 U/μL XbaI (NEB, #R0145T) and 4 μl 5 U/μL I-SceI (NEB, #R0694L) in a 50 μL final volume for 3 hours at 37­ °C. Digested DNA was column-purified (QIAGEN, QIAquick PCR purification kit #28104). UMI-amplicons: To eliminate I-SceI sites that were not cut during DSB induction and limit the size of PCR extension, 2 μg genomic DNA from mESCs (both, with DSB induction and control without the induction) were digested using 1 μl 100 U/μL XbaI (NEB, #R0145T) and 4 μl 5 U/μL I-SceI (NEB, #R0694L) in a 50 μL final volume for 3 hours at 37­ °C. Digested DNA was column-purified (QIAGEN, QIAquick PCR purification kit #28104).  UMI-LA 500 ng of genomic DNA obtained after I-SceI/XbaI digestion was used as a template in a 50 μL of Q5 DNA polymerase reaction mix (NEB, M0491S), using 50 nM of a UMI-containing primer U1 in the following cycling conditions: 98°C for 4 min; 98°C for 30s, 60°C for 1 min and 72°C for 1 min (50 cycles); and 72°C for 5 min. Products of linear amplification were purified using Agencourt AM Pure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. Linear amplification was repeated 4 times for every sample to account for the technical variability. UMI-PCR: 500 ng of genomic DNA obtained after I-SceI/XbaI digestion was used as a template in a 50 μL of Q5 DNA polymerase reaction mix (NEB, M0491S), using 50 nM of primers U1 and U2 in the following cycling conditions: 98°C for 1 min; 98°C for 20s, 60°C for 1 min and 72°C for 4 min (6 cycles); and 72°C for 10 min. Products of UMI-PCR were purified using Agencourt AM Pure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. UMI-PCR was repeated 4 times for every sample to account for the technical variability.