alias: GSM3677629
title: GSM3677629: Rbp4Cre_Rep4_TRAP - 1; Mus musculus; OTHER
library_construction_protocol: RNA was extracted from lysates with Qiagen RNeasy kit using manufacterer's protocols SRT libraries were prepared from cortex RNA samples. Prior to library preparation, cortex samples were dissected into 10 separate pieces, from which RNA was independently isolated with the manufacturer's protocol (Qiagen RNeasy kit, Germantown, MD, USA). This allows for identification of up to 10 independent insertion events into any TTAA site, given that these insertions occur in spatially separate samples. From these RNA samples, transposon sequencing libraries were generated with our bulk SRT protocol (Moudgil et al., in revision). In brief, RNA samples were first reverse transcribed, from which self-reporting transcripts, including flanking genomic sequences, were amplified via PCR. These amplicons were then tagged with universal Illumina sequencing overhangs, with separate indexes for libraries from each dissected piece, allowing for 10 'barcodes' per sample and sequenced on Illumina HiSeq 2500 or NextSeq 500 platforms (Illumina, San Diego, CA, USA). SRT calling card DNA was extracted from lysates with Qiagen DNeasy kit using manufacterer's protocols BrokenHeart libraries were prepared from cortical DNA samples, as previously described (Wang et al., Genetics, 2012). A set of 20 individually barcoded BrokenHeart transposons were pooled and packaged into AAV. Thus, independent insertions into the same TTAA can be uniquely identified via barcode, removing the need to dissect and process tissue samples in separate pools as with SRT. Extracted DNA was self-ligated, amplified with inverse PCR, and sequenced with the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA). DNA calling card
identifiers.namespace: GEO
identifiers.id: GSM3677629