GSM6793003: HepG2.LTR.growth.B1; Homo sapiens; OTHER
DNA was isolated with TIANamp Genomic DNA Kit (Tiangen) 1 μg of genomic DNA was used as the template for a linear PCR in a 50 μl reaction system containing 1x Taq Buffer, 2.5 U Taq DNA polymerase (Tiangen), 4 μl of 2.5 mM dNTP mix (Takara), and 0.3 μM biotinated primer 3-LTR_prime (5'-biotin-GCTCAACTGGTACTAGCTTGTAGCACCATCC-3'), which anneals to the 3' LTR region of the lentivirus vector. 50 cycles of linear PCR were performed with 2.5 U Taq DNA polymerase (Tiangen) added at the beginning and immediately after 25 cycles of the reaction. PCR conditions were as follows: 94℃ for 5 min; 50 cycles of 94℃ for 30 s, 55℃ for 30 s and 72℃ for 30 s; 72℃ for 5 min. After the linear amplification, the PCR products were mixed with 10 μl of BeaverBeadsTM Streptavidin (Beaver) and incubated for 2 h at room temperature with shaking (400 rpm) in a metal bath. Beads were then collected with a magnetic stand and washed with 300 μl Binding/Wash buffer (10 mM Tris-HCl (pH 7.5), 1 M NaCl, 1 mM EDTA, 0.1% Tween-20) for 6 times, and with 65°C H2O for twice. For the second strand synthesis, beads were resuspended in a 24 μl reaction volume containing 1x Klenow Fragment Buffer (NEB), 2 μl of 2.5 mM dNTP mix (Takara), 6.25 μM P5_N6 primer (5'-CTACACGACGCTCTTCCGATCTNNNNNN-3') and pre-incubated at 15℃ for 20 min. Then, 2 U of Klenow polymerase (NEB) were added and the mixtures were incubated at the following conditions: slow ramp at 0.5℃ per minute from 15℃ to 25℃ followed by 30 min incubation; then slow ramp at 0.5℃ per minute to 37℃ followed by 1 h incubation. Beads were captured with a magnetic stand and washed gently with 300 μl of 4℃ water. To construct the library for NGS, nested PCR was performed. First, beads were resuspended in a 25 μl PCR reaction volume containing 1x Taq Buffer, 1.25 U Taq DNA polymerase (Tiangen), 2 μl of 2.5 mM dNTP mix (Takara), 0.5 μM of primer P5 (5'-CTACACGACGCTCTTCCGATCT-3') and 0.5 μM of primer 3-LTR_Nest (5'-CCTGGTGTGTAGTTCTGCCAATCAG-3'). PCR conditions were as follows: 94℃ for 3 min; 20 cycles of 94℃ for 30 s, 55℃ for 30 s and 72℃ for 30 s; 72℃ for 7 min. Second, 5 μl of the PCR products from the previous step were used for the next round of PCR in 50 μl volume containing 1x Taq Buffer, 2.5 U Taq DNA polymerase (Tiangen), 4 μl of 2.5mM dNTP mix (Takara), 0.4 μM of primer Illumina_P5 (5'-AATGATACGGCGACCACCGAGAtctACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and 0.4 μM of primer Illumina_P7-3LTR (5'- CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTTGTGTGTGGTAGATCCACAG-3'). PCR conditions were as follows: 94℃ for 3 min; 15 cycles of 94℃ for 30 s, 55℃ for 30 s and 72℃ for 30 s; 72℃ for 7 min. PCR products were purified with 1.2x volumes of VAHTS DNA Clean Beads (Vazyme) to a final volume of 21 μl. The concentration of PCR products was measured by Qubit 3.0 fluorometer using Equalbit dsDNA HS Assay Kit (Vazyme). To achieve a better coverage, five libraries were prepared in parallel from 1 μg of genomic DNA and pooled together.