title: GSM5915889: Exp7 Spleen on 2738.AAV.PGK.GFP (LTR35.LC48) rep 2; Homo sapiens; OTHER
library_construction_protocol: gDNA was sheared using a Covaris E220 Ultrasonicator (Covaris Inc.), generating fragments with a target size of 1,000 bp. The fragmented DNA was subjected to end repair, 3' adenylation and ligation (NEBNext® Ultra™ DNA Library Prep Kit for Illumina, New England Biolabs) to custom linker cassettes (LC) (Integrated DNA Technologies). LC sequences containing 8-nucleotide barcode for sample identification. Ligation products were subjected to 35 cycles of exponential PCR with primers complementary to different regions of the AAV or IDLV genomes and to the LC. For each set of AAV or IDLV specific primers, the procedure was performed in technical replicates (n = 2-3) using 80-100 ng of sheared DNA/each. Next, ten additional PCR cycles were done to include sequences required for sequencing and a second 8-nucleotide DNA barcode. PCR products were quantified by qPCR using the Kapa Biosystems Library Quantification Kit for Illumina, following the manufacturer's instructions. qPCR was performed in triplicate on each PCR product diluted 10:3, and the concentrations were calculated by plotting the average Ct values against the provided standard curve. The amplification products were sequenced by Illumina Next/Novaseq platforms (Illumina).