GSM7776022: Amplicon sequencing of untreated control [Amp-LAMA3-UT]; Homo sapiens; OTHER
Genomic DNA was extracted using the ReliaPrep™ Blood gDNA Miniprep System kit (Promega) according to the manufacturer's protocol 80 ng of genomic DNA was used as a template for amplification via a 30-cycle PCR using Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, NEB). PCR fragments were purified using the QIAquick PCR Purification Kit (QIAGEN), subjected to Sanger sequencing (Eurofins Genomics) and analyzed using DECODR.57 Alternatively, purified PCR products were pooled and prepared for NGS using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB), and sequenced on Illumina HiSeq or NovaSeq platforms with 2× 150 bp read length (GENEWIZ, Azenta Life Sciences). Sequencing was outsourced to service NGS provider Genewiz (part of Azenta Life Sciences) where it was performed on a Illumina NovaSeq6000 device collecting 2x150 bp paired-end reads.