title: GSM8792849: No Pt-drug treatment on ICR-191 treated CHO rep1; Cricetulus griseus; OTHER
library_name: GSM8792849
library_construction_protocol: Pt-seq: Genomic DNAs were extracted using Universal Genomic DNA Kit (CWBIO, CW2298) following manufacturer's protocol. Target-seq: Total RNA of CHO cells was extracted by Total RNA Isolation Kit (Vayzme, RC112) and then reverse transcription was performed using 1st Strand cDNA Synthesis Kit (Vayzme, R312). Pt-seq: Briefly, 500-1000 ng genomic DNA was fragmented by COVARIS S220 Focused-ultrasonicator (Covaris, Inc.) to generate DNA fragments with a medium size of 300 bp. DNA fragments with 1 ng model sequences were performed for library preparation by using End Repair/dA-Tailing Module (NEB, E7546). Adaptors with modifications used in Pt-seq were synthesized by Sangon Biotech, Shanghai and annealed by gradual cooling process. Ligation Module (NEB, E7595) was used to perform restriction-sites-contained-adaptor ligation. Then DNA fragments were denatured and placed on ice. Non-immunoprecipitated DNA was saved as input and stored at -20°C. Generally, 1 μg anti-Pt modified DNA antibodies (Abcam, ab103261) were added into IP buffer and incubated with the processed DNA fragments overnight at 4°C with gentle rotation. Dynabeads Protein G (Thermo Fisher, 10004D) were pre-washed and blocked with 0.1% BSA/PBS and then incubated with samples for 2 hours at 4°C with gentle rotation50. The immunoprecipitated DNA-antibody-Dynabeads complexes were washed three times with IP buffer. The immunoprecipitated DNA was eluted by Proteinase K (NEB, P8107S) with freshly made elution buffer at 56°C for 2 hours and then purified. Then un-ligated DNA was removed by RecJf (NEB, M0264) and Exonuclease I (NEB, M0293). Then the primer extension was performed using Q5 High Fidelity DNA Polymerase (NEB, M0544) and redundant primer was removed by Exonuclease I (NEB, M0293). DNA fragments were denatured and placed on ice. Second adaptor ligation was performed on sing strand DNA samples using T4 DNA Ligase (NEB, M0202) with PEG8000 at 16°C for overnight. PCR amplification was performed using Q5 High Fidelity DNA Polymerase (NEB, M0544) and DNA was amplified for 2 cycles at first time. The PCR products were cleaned up with 1.2× AMPure XP beads (Beckman Coulter). Restriction endonuclease digestion was performed using SbfI (NEB, R3642) at 37°C f. Target-seq: The cDNA was diluted and amplified with primers specially designed for HGPRT gene containing the paired adaptor sequences (Supplementary Table 1) using Q5 High Fidelity DNA Polymerase (NEB, M0544) for 5 cycles. The PCR products were purified with 1× AMPure XP beads (Beckman Coulter) and then the second round of amplification was performed with different index primers for about 25 cycles. The PCR products were purified with 0.8× AMPure XP beads (Beckman Coulter). Final library quality was measured as described before. Libraries were sequenced on and MGISEQ-2000 platform to generate 150 bp paired-end reads.