Pfuturbo cx hotstart dna polymerase

RNA was extracted from RNP preparations using the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions, omitting the cell lysis step and including on-column DNase treatment. RNA was quantified using a Nano-Drop spectrophotometer and diluted to 0.5 μg/μl. RT-PCR was carried out on 0.5 μl of purified RNA, using MMLV reverse transcriptase (Promega, Madison, WI) and 0.8 μM LEAP adapter (5np1) at 42°C for 30 min. Reactions containing 100–300 ng of rA3A or rA3A_C106S protein in 10% glycerol were included in the MMLV RT reaction. For control reactions containing ‘heat-killed’ rA3A, rA3A samples were incubated at 100°C for 15 min prior to inclusion in the MMLV RT reaction. Following the MMLV RT reaction, reaction mixtures were incubated at 100°C for 15 min to denature rA3A protein. An aliquot (0.5 μl) of the MMLV-RT reaction product was PCR amplified with PfuTurbo C_x Hotstart DNA polymerase (Agilent) using the following cycling conditions: 95°C for 2 min, followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C, with a final extension time of 7 min at 72°C. PCR products were visualized on 2% agarose gels.

WGBS of Atf7ip and Zmym2 KO mESCs was performed as described previously^{64 (link)}. In short, 10 µg genomic DNA was sonicated to a length of approximately 400–500 bp. For each sample, 2 µg sheared genomic DNA was mixed with 10 ng equimolar pooled sonicated methylated phage T7 and unmethylated phage Lambda DNA. Adapter-ligation was carried out with the NEBNext Ultra II kit (NEB E7645L) using methylated adaptors (NEB, E7535S), before bisulfite conversion using the Qiagen Epitect bisulfite conversion kit, according to the manufacturer’s instructions. After conversion, libraries were amplified for 10 cycles using the Pfu TurboCx Hotstart DNA polymerase (Agilent) and the NEB dual index primers (NEB, E7600S). PCR reactions were run with the following parameters: 95 °C for 2 min, 98 °C for 30 s, followed by 10 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 3 min, ending with 5 min at 72 °C. The PCR reactions were cleaned up using 1.2× AMPure XP beads (Beckman Coulter) and eluted in 20 µl EB buffer (Qiagen). Library quality was checked on an Agilent TapeStation and sequencing was done on an Illumina NovaSeq 6000 machine.

A total amount of 5–10 μg genomic DNA was mixed with 25 ng lambda DNA and sonicated to 200–500 bp, followed by end repair and dATP adding with the homemade Kit. Next, methylated adapters (synthesized by ThermoFisher Scientific) were ligated to the sonicated DNA. Ampure beads (Vazyme) were used to remove <200 bp fragments. Bisulfite treatment was performed using the EZ DNA Methylation-Gold TM Kit (ZYMO research, USA). After bisulfite conversion, the single-stranded, uracil-containing DNA was subjected to 10–12 cycles of PCR reaction with Illumina TruSeq PCR primers and 2.5 U of Pfu Turbo C_x Hotstart DNA polymerase (Agilent Technologies) to recover enough DNA for sequencing. The sequencing reads were aligned to mm9 using BSMAP with parameters –r 0 –w 100 –v 0.1 –A AGATCGGAAGAGC. Multiple mapped reads and PCR duplicates were removed. After mapping, those reads with total CG coverage less than 5 within 200 bp were removed. The methylation level was calculated using methylated CpG versus total CpG in each bin. The file with DNA methylation levels for each 200 bp bin was used for further analysis. For the boxplots about DNA methylation, we calculated the average DNA methylation levels of each region for each kinds of gene elements. The genomic information of maternal DMRs (n = 20) and paternal DMRs (n = 3) used in the WGBS analysis were shown in Table S4.

RRBS assay was performed as previously described^{75 (link)}. Briefly, 20 ng of genomic DNA were digested with Msp1 restriction enzyme (NEB, R0106L), DNA fragments were end-repaired and A-Tailed using Klenow fragment (3′−5- exo-) (NEB, M0212L). The DNA fragments were then ligated to illumina adaptors (Illumina, PE-940-2001) using T4 ligase (NEB, M0202M) and then size selected using AMPure XP beads (Beckman Coulter Genomics, A63881). The samples were then subjected to two consecutive bisulfite conversions using EpiTect Bisulfite Kit (QIAGEN, 59104) and PCR using PfuTurbo Cx hotstart DNA polymerase (Agilent Technologies, 600412). The RRBS libraries were sequenced by Illumina HiSeq 2000 platform.