GSM6807488: Mouse-L-2.Nova; Mus musculus; OTHER
DNA was isolated with TIANamp Genomic DNA Kit (Tiangen) The biochemistry part of 3' ends tailing was based on the SSiNGLe-ILM protocol from our previous study (PMID: 31862872). Briefly, 100 ng of the purified DNA was denatured at 95℃ for 5 min in 19 µl volume containing 2 µl of 10x TdT buffer and 2 µl of 2.5 mM CoCl2 followed by rapid snap-cooling on ice. TdT (5 units, NEB) and 2 µl of 10 mM dATP (Takara) were then added to denatured DNA to a total volume of 22 µl and incubated at 37°C for 30 min. To block free 3′-OH ends, samples was mix with 2 µl of 10 mM ddNTP (Roche) and incubated at 37℃ for additional 30 min, followed by enzyme of VAHTS DNA clean beads to a final volumn of 15 μl and mixed with 2 µl of 10x Taq buffer (Tiangen), 1.6 µl of 2.5 mM dNTP mix (Takara), 1 µl of 10 µM of polyT primer (a chimeric DNA-RNA oligo-d(T)50-r(T)3 oligonucleotide) and 1 unit of Taq DNA polymerase (Tiangen). The amplification conditions were as follows: initial denaturation at 94℃ for 3 min, followed by 10 cycles of denaturation at 94 °C for 30 s, annealing at 55℃ for 30 s and extension at 72℃ for 30 s. The PCR products were purified with 2x volumes of VAHTS DNA clean beads and tailed with dCTP (Takara) with the same procedure used for polyA tailing, followed by blocking of 3'-OH ends as described above. The tailing products were purified with 2x volume of VAHTS DNA clean beads and all purified DNA was PCR-amplified with P5_10N10G (CTACACGACGCTCTTCCGATCTAGTTGCGGATGGGGGGGGGGHN) and P7_T12 (GTTCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTVN) primers with the following amplification conditions: initial denaturation at 94℃ for 3 min; 1 cycle of denaturation at 94℃ for 30 s, annealing at 55℃ for 1 min and extension at 72℃ for 1 min; 1 cycle of denaturation at 94℃ for 30 s, annealing at 37℃ for 1 min and slow ramp at 2℃ per minute to 72℃; 30 cycles at 94℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s; final extension at 72℃ for 7 min. The amplified DNA was purified with 2x volumes of VAHTS DNA clean beads to a final volumn of 17 μl of water, and 4 µl PCR products were used as the template for the second round of nested PCR with lIlumina_P5 (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and primer Illumina_P7 (CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) as primers under the following conditions.: 94℃ for 3 min; 6 cycles of 94℃ for 30 s, 55℃ for 30 s and 72℃ for 30 s; 72℃ for 7 min. PCR products were purified with 2x volumes of VAHTS DNA Clean Beads.