GSM6604922: IMPB10_4B_MES_LN; Mus musculus; OTHER
DNA isolation of tissue samples was performed according to manufacturer's instructions using the Qiagen DNeasy Blood & Tissue Kit. DNA samples were sheared with a Covaris AFA sonicator to a mean fragment length of 250 bp. The fragmented DNA was then end-repaired, A-tailed and a splinkerette adapter was ligated to each DNA end. For the 5' and 3' transposon end, subsequent steps (amplification and sequencing of transposon-genome junctions) were conducted separately. The specific structure of the splinkerette adapter (Y-shaped design with a template and a hairpin strand) ensures that only transposon-genome junction fragments (and not genomic fragments without transposon insert) can be amplified in the first PCR step (which was conducted with transposon- and splinkerette-specific primers). Afterwards, a second nested PCR step was performed for further amplification, barcoding of samples and extension with Illumina flow cell binding sites P5 and P7. Samples were equimolarly mixed and the library pool was again quantified. Libraries were sequenced on the Illumina MiSeq sequencer (75 bp, paired-end).