GSM8489051: delta_T0_Repica2; Homo sapiens; OTHER
Genomic DNA was extracted using NucleoSpin Blood kits from Macherey-Nagel, following the manufacturer's instructions. Total genomic DNA (gDNA) concentration was measured, and the corresponding number of PCR reactions were assembled to use 10 µg gDNA per reaction (~100 reactions per sample). PCR reactions were prepared with NEBNext Ultra II Q5 MasterMix, gDNA, Forward (aatgatacggcgaccaccgaGATCTACACGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAgcacaaaaggaaactcaccct), and Reverse (caagcagaagacggcatacgaGATCGACTCGGTGCCACTTTTTC) primers. Note that the underlined sequence is an Illumina barcode and varies for each sample. The following PCR conditions were used: 1. Initial denaturation for 30 seconds at 98ºC, 2. Denaturation at 98ºC for 10 seconds, 3. Annealing/extension at 65ºC for 75 seconds, 4. Repeat steps 2-3 twenty-one more times, 5. Final extension at 65ºC for 5 minutes. The PCR product was purified using SPRIselect Reagent (Beckman Coulter). Run-type single end 50, Index length: 1x6, Sequencing primers: Read 1, custom primers (gtgtgttttgagactataaGtatcccttggagaaCCAcctTGTTG); Read 2, standard Illumina index, Spike in 20% PhiX to maintain diversity  primer