Kod multi epi

Genomic DNA was purified using DNeasy Blood & Tissue Kit at indicated days of differentiation. Bisulfite conversion of genomic DNA was performed with 500 ng of genomic DNA using EpiTect Plus DNA Bisulfite Kit (QIAGEN, 59124) as the manufacture's manual. Target sequences were PCR-amplified using KOD -Multi & Epi- (TOYOBO, KME-101) with specific primer sets targeting the coding strands after the bisulfite conversion, which were designed with MethPrimer (https://www.urogene.org/methprimer/) (62 (link)). PCR products were subcloned into pCR-Blunt II TOPO vector (Thermo Fisher, 451245) or pCR4-Blunt TOPO vector (Thermo Fisher, 450031). Plasmids were isolated by boiling transformed E. coli for 1 min in buffer containing 0.7 mg/ml lysozyme, 10 mM Tris–HCl, pH 7.5, 63 mM EDTA, 2.5 M LiCl, and 4% Triton X-100 with immediate cooling on ice and purified by isopropanol precipitation. The inserts were PCR-amplified by M13 forward and reverse primers using KOD-Plus Neo (TOYOBO, KOD-401) and subjected to Sanger sequencing. All primers for DNA methylation analysis are listed up in Supplementary Table S9. Sequence data was analyzed using QUantification tool for Methylation Analysis (QUMA) (http://quma.cdb.riken.jp/) (63 (link)).

Total RNA was extracted from fin clips or paired testes with RNAiso Plus (Takara) and treated with TURBO DNase (Ambion). Testis cDNA was prepared from 1 μg of total RNA with a PrimeScript RT-PCR kit (Takara) using oligo dT primers. qPCR was performed with LightCycler 480 SYBR Green I Master on a LightCycler 480 system (Roche) with the default settings, except that annealing was performed at 58°C. For the cloning of sycp2 cDNA and the analysis of sycp2 splicing at exons 8–9, PCR was performed using KOD -Multi & Epi- (Toyobo) on cDNA from five testes each of sycp2^+/+ and sycp2^its/its males. All primers used in this study are listed in S3 Table.

A 2.2-kb DNA fragment encompassing the human PD-1 promoter was cloned into pBluescriptII and incubated with NTP-GM or CpG methyltransferase (M.SssI) (New England BioLabs, Ipswich, MA) in a reaction buffer of 50 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂ and 1 mM DTT. After incubation for 3 h at 37 °C, the DNA was subjected to a Cells-to-CpG Bisulfite Conversion Kit (Applied Biosystems, Foster City, CA), and the targeted loci (CR-C and CR-B) were amplified by PCR using KOD-Multi&Epi-® (Toyobo, Osaka, Japan), followed by sequencing. Primers were designed to amplify an approximate 250–300-bp fragment of the CpG islands in CR-C and CR-B. Nucleotide sequences of primers for CR-C were 5′-GGGAGTGGTTTTTTGTTTATAAA-3′ (forward primer) and 5′-AAAATCCAATACCTAAACCTAACTAAC-3′ (reverse primer), whereas those for CR-B were 5′-TTAATTTGATTTGGGATAGTTTTTTTT-3′ (forward primer) and 5′-CCCTCCAAACCCCTAACTCTAAAAC-3′ (reverse primer). Methylation frequency was measured by using Quantification Tool for Methylation Analysis.^{23 (link)}MOLT-4 cells, PBMCs and NK cells were seeded onto a 96-well plate at a density of 2.0 × 10⁵ cells/mL. After 24 h, 1–20 nM NTP-GMs were added to each well once a day for 5 days. On the day following the last addition of the proteins (day 6 after the initial addition), the cells were subjected to analysis.

The 16 purified gDNAs were submitted to a service provider, Apical Scientific Sdn. Bhd. for 16S rRNA gene amplification and amplicon sequencing. The 16S rRNA gene was amplified by targeting the V3–V4 region using locus-specific bacterial 16S rRNA V3–V4 (Caporaso et al. 2012 (link)) primer sets with overhang adapters Forward: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3’ and Reverse: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-3’. All PCR reactions were carried out using KOD -Multi & Epi-® (Toyobo), following manufacturer’s instructions. Dual indices were attached to the amplicons PCR using Illumina Nextera XT Index Kit v.2, following manufacturer’s instructions. The quality of the libraries was measured using an Agilent Bioanalyzer 2100 System by Agilent DNA 1000 Kit and fluorometric quantification was performed using a Helixyte GreenTM Quantifying Reagent. Finally, the libraries were normalised and pooled, followed by sequencing with the MiSeq platform using 300 paired-end sequencing, according to manufacturer’s instructions (Illumina Inc., San Diego, CA, USA).