title: GSM2356422: ENHANCER_gSNP_IL2RA+_24hrs_D1; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was extracted from sorted cells using QuickExtract (Epicentre). PCR amplicons from the edited regions were prepared for next-generation sequencing using a nested PCR strategy. The outer PCR (PCR1) generates a 600-700 bp amplicon using primers outside the region matching the HDR donor template. The inner PCR (PCR2) generates the ~250 bp amplicon to be sequenced. For PCR1, genomic DNA from 5,000-10,000 sorted cells was amplified using PrimeStar GXL DNA Polymerase (Clontech, Cat. No. R050B) according to the following protocol: 98 °C, 2’; 30 cycles at 98 °C, 10”, 55 °C, 15”, 68 °C, 45”. PCR1 products were purified using SPRI beads and quantified using the Qubit dsDNA HS Assay Kit (Thermo). The purified products of PCR1 (20 ng/sample) were used as templates for PCR2 and amplified using PrimeStar GXL DNA Polymerase according to the following protocol: 98 °C, 2’; 10 cycles at 98 °C, 10”, 60 °C, 15”, 68 °C, 20”. PCR2 products were purified and quantified as done for PCR1. Indexed sequencing libraries were prepared with the TruSeq Nano DNA HT Library Prep Kit (Illumina, Cat. No. FC-121-4001) using 100 ng of purified PCR2 product. Libraries were pooled and 150 bp paired-end sequencing was performed with a MiSeq sequencer (Illumina). Amplicon Sequencing