GSM6796744: AAV-mRNA, rep1; Homo sapiens; OTHER
Genomic DNA from CAR-T cells was extracted using QIAGEN's Blood Mini Kit. First, gDNA was fragmented using NEB Ultra II FS Enzyme Mix. Then, Splinkerette adaptors were annealed by heating to 98C for 10 min and then cooling to room temp. Annealed Splinkerette adaptors were ligated to fragmented DNA using NEBNext Ultra II Ligation Master Mix. Library fragments were size selected for an approximate insert size of 150-200bp using SPRIselect magnetic beads. A two step PCR was then performed: the first PCR to amplify integrated transposon sequences (18 cycles) and the 2nd PCR to attach sequencing indeces (8 cycles). The libraries for each sample were quantified by qPCR, diluted to 2nM, and then pooled for sequencing by Miseq. insertion site mapping