organism: Homo sapiens
description: Protocols: Cells were transfected with a plasmid-borne L1.3 element carrying a NeoR-based retrotransposition cassette and a hygromycin-resistance gene in the vector backbone. Transfected cells were selected with hygromycin (but not by G418) for 10 days. Genomic DNA was extracted using a QiaAmp DNA Blood mini kit (Qiagen). ATLAS-seq-neo libraries were prepared by random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, digestion of contaminating plasmid DNA with BamHI, and suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence. The primers used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016), and are fused to Ion Torrent-specific A and trP1 primers. Libraries are oriented with reads directed from the flanking sequence to the L1 3' end (reads have the following structure: 5'-A-barcode-linker-flank-poly(dT)-L1-trP1-3'). This library construction protocol used suppression PCR reactions performed in solution. In this set of experiments, linker-ligated DNA was digested with BamHI, which cuts downstream of L1 polyA site in the plasmid backbone, to prevent amplification from the plasmid and enrich for bona fide insertions in the genomic DNA.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-04-03T13:14:34Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-02-01T16:10:31Z
attributes.tag: External Id
attributes.value: SAMEA5315641
attributes.tag: INSDC center name
attributes.value: Universite Cote d'Azur, Inserm, CNRS
attributes.tag: INSDC first public
attributes.value: 2019-04-03T13:14:34Z
attributes.tag: INSDC last update
attributes.value: 2019-02-01T16:10:31Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7644:SH09_RD
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HeLa-S3
attributes.tag: common name
attributes.value: human
attributes.tag: growth condition
attributes.value: Hygromycin
attributes.tag: replicate
attributes.value: 9
attributes.tag: sample name
attributes.value: E-MTAB-7644:SH09_RD
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female