GSM5277227: Cas9 Day 8 -DOX; Homo sapiens; OTHER
Genomic DNA was extracted using the phenol-chloroform method. The genomic DNA were digested with RNase A (OMEGA) to remove RNA contamination (In this study, 50 µg RNase A worked well to digest the RNA contamination in 100～200 µg genomic DNA after incubating in 37 ℃ for 30 min). PCR-free library was prepared using MGIeasy FS PCR-free DNA library Prep kit following the manufacturer's instruction. Briefly, measure the concentration of purified PCR products using Qubit 4 TM fluorometer (Invitrogen) and dilute the concentration of each sample to 10 ng/µl. 10 µl diluted PCR product was mixed with an A-Tailing reaction which contained A-Tailing enzyme and buffer, incubated at 37°C for 30 minutes then 65°C for 15 min to inactive the enzyme. Then the A-Tailed sample was mixed with PCR Free index adapters (MGI.), T4 DNA Ligase and T4 ligase buffer to add index adapter at both 3' and 5' ends of PCR products.  The reaction was incubated at 23°C for 30 min and then purified with XP beads. Then denature the PCR products to be single-strand DNA (ssDNA) by incubating at 95 °C for 3 min and keep on 4 °C for the subsequent step. Transform the ssDNA to be circles using cyclase (MGI) at 37 °C for 30 min and then digested to remove linear DNA using Exo enzyme at 37 °C for 30 min. Purify the products again by XP beads and assay the concentration of library by Qubit 4 TM fluorometer. Liraries were sequenced with pair-ended 150 bp deep-sequencing on a MGISEQ-2000 sequencing platform