Kapa hifi hotstart uracil readymix pcr kit

We performed WGBS for Pa, Ra, and Rb samples. Purified genomic DNA of each sample was mixed with 0.1% lambda DNA, and sonicated using a Covaris S220 instrument (Woburn, MA, USA). End-repair, dA-tailing, and ligation were performed using the NEBNext End Repair Module, dA-Tailing Module, and NEB T4 ligase, respectively. Subsequently, DNA fragments were ligated with methylated adaptors. Size selection was performed by gel extraction to obtain DNA fragments larger than 200 bp. The adaptor-ligated DNA was treated with sodium-bisulfite using the EZ DNA Methylation-Gold™ Kit (Zymo, Irvine, CA), and subsequently amplified using KAPA HiFi HotStart Uracil + ReadyMix PCR Kit (KAPA Biosystems, Roche, Wilmington, MA) with 10 cycles. Paired-end sequencing of 2 × 100 bp was performed on the Illumina HiSeq 2000 platform at the core facility of the Beijing Institute of Genomics, CAS.

Gene-specific DNA methylation was assessed by a next generation sequencing-based targeted bisulfite sequencing, according to previously published method [18 (link)–21 (link)]. In brief, primers were designed using the online MethPrimer software and listed in Table 2. 200 ng of genomic DNA was converted using the ZYMO EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) and were used as templates for PCR amplification with 35 cycles using KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA). For each sample, PCR products of multiple genes were pooled equally, 5’-phosphorylated, 3’-dA-tailed and ligated to barcoded adapter using T4 DNA ligase (NEB). Barcoded libraries from all samples were sequenced on Illumina Nova6000 platform using a 150×2 paired-end sequencing protocol.

Bisulfite treated DNA and KAPA HiFi HotStart Uracil + ReadyMix PCR Kit (KAPA biosystems) was used to set up the system for amplification. The bisulfite PCR primers for LKB1 promoter were designed on MethPrimer website (Fig. 1e) [24 (link)]. The primer sequences are listed as follow: Forward 5′- GAG GAT GAT TTA GTA TTG AAA AGT-3′; Reverse 5′- AAC AAC AAA AAC CCC AAA AA-3′, product size: 259 bp (containing 21CpG sites). The reaction was performed under 95 °C for 5 min, followed with 39 cycles of 98 °C for 20s, 59 °C for 15 s, and 72 °C for 1 min; and then 72 °C for 10 min. The product was uploaded to 1.5% agarose gel and the purification was done by TIANgel Mini Purification Kit (TIANGEN). The purified product was ligated to pGM-Simple-T Fast Vector (TIANGEN) by T4 DNA ligase (NEB). The ligated vector was transfected into DH5α competent cells. LB agar plate was used for monoclonal selection. Sanger sequencing was sent to Sangon Biotech. Each sample was required at least 10x coverage. Sequences was aligned to reference LKB1 promoter sequence, and visualized by BiQ analyzer [25 (link)].

The next-generation sequencing-based BSP was performed to examine the gene DNA methylation following the previously reported method [19 (link)]. In brief, genomic DNA was extracted from ovarian cancer cells using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Bisulfite sequencing (BS) was utilized to determine the DNA methylation status of the CpG islands of the promoter region of UBE2C. The bisulfite conversion of genomic DNA was performed using the ZYMO EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. BSP primers of UBE2C were designed using the online MethPrimer software (http://www.urogene.org/methprimer/; accessed 25 June 2021) and listed in Table S1. Bisulfite-treated genomic DNA was used to amplify the CpG islands of the UBE2C promoter, which contains 47 CpG sites, using KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA). Amplified PCR products were pooled in equal volumes, 5’-phosphorylated, 3’-dA-tailed, and ligated to a barcoded adapter using T4 DNA ligase (NEB). The barcoded libraries were sequenced on an Illumina platform.

Genomic DNA of liver was extracted by Quick-DNA Universal Kit (ZYMO Research, USA), and was broken into 400–500 bp fragments using sonication(Qsonica q700). End repair and adaptor ligation were performed by the following 2 kits: NEBNext^® Ultra™ II DNA Library Prep with Sample Purification Beads (NEB, USA) and NEBNext^® Multiplex Oligos for Illumina^® (Methylated Adaptor, Index Primers Set 1) (NEB, USA). Adaptor-ligated DNA was cleaned up without size selection. Bisulfite conversion was performed by MethylCode™ Bisulfite Conversion Kit (Invitrogen, USA). PCR amplification of adaptor-ligated and bisulfite converted DNA were performed by KAPA HiFi Hotstart Uracil + ReadyMix PCR Kit (KAPA Biosystems, USA). The 1.8pM library and 20pM PhiX control (Illumina, USA) were prepared following the NextSeq System Denature and Dilute Libraries Guide using NextSeq 500/550 v2 Kit (high output 150 cycle) (Illumina, USA). WGBS was performed in the Center for Epigenetics & Disease Prevention, Institute of Biosciences & Technology, Texas A&M University (Houston, TX, USA). WGBS data is now accessible at the BioProject database (BioProject ID: PRJNA777555).