Nucleospin tissue kit

Genomic DNA was extracted with a NucleoSpin Tissue kit (Takara Bio, Shiga, Japan) and subjected to quality control by agarose gel electrophoresis. The DNA obtained was quantified with a Synergy H1 (BioTek, Winooski, VT, USA) and QuantiFluor dsDNA System (Promega, Madison, WI, USA), and sequenced using a NextSeq platform (Illumina, Inc., San Diego, CA, USA) using a 2 × 151-bp paired-end approach. After quality control on raw reads was performed using Sickle (https://github.com/najoshi/sickle), the reads were assembled de novo using SPAdes version 3.10.1. Gene annotation was determined with Prokka version 1.11. The draft genome was subjected to in silico analyses, such as MLST using MLST version 2.0 [20 (link)] and detection of antibiotic resistance mechanisms using ResFinder version 3.1 [21 (link)], ARG-ANNOT [22 (link)], and CARD [23 (link)].

DNA was extracted with the NucleoSpin Tissue kit (Takara, Japan). Bisulfite conversion of fragmented purified DNA was performed using the EpiTect Bisulfite Kit (Qiagen). Bisulfite-modified DNA was amplified by PCR with BGS primers as described in Table S2. Amplified PCR products were cloned into the TA cloning vector pT7-Blue and sequenced. The analyzed murine KCC3 promoter region is presented in Figure S6A, and 5 CpGs (CpGs 1–5) were included in the region.

The provirus copy number per diploid human genome in lentivector-transduced (LV-lcoET3) cells was determined using the Lenti-X Provirus Quantitation Kit (Takara, Mountain View, CA) according to the manufacturer’s protocol. Briefly, genomic DNA was isolated from transduced and non-transduced cells using the NucleoSpin Tissue kit (Takara, Mountain View, CA). Serial dilutions were made with genomic DNA, and qPCR amplification was carried out along with a standard curve derived from serial dilutions of calibrated provirus control template. The raw Ct values of the sample were correlated to the standard curve to determine the provirus copy number per cell. A correction coefficient was also incorporated to compensate for the different PCR sensitivities to amplifying provirus control template only vs. provirus sequence integrated in genomic DNA.

We extracted DNA from cell lines by using Nucleospin tissue Kit (Takara Bio Inc., Shiga, Japan), and DNA from surgically obtained formalin-fixed paraffin embedded (FFPE) tissue by using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) (23 (link), 32 ). PCR was performed to amplify fragment of H3F3A gene or HIST1H3B gene including the mutational region of H3K27M. Primer sequences for the H3F3A were 5′- GAT TTT GGG TAG ACG TAA TCT TCA−3′ (forward) and 5′- TTT CCT GTT ATC CAT CTT TTT GTT−3′ (reverse) and for the HIST1H3B were 5′- GGG CAG GAG CCT CTC TTA AT−3′ (forward) and 5′- ACC AAG TAG GCC TCA CAA GC−3′ (reverse) as described previously (33 (link)). Polymerase chain reaction (PCR) products were electrophoresed on agarose gel and target DNA fragments were excised from the gel and purified by using Wizard SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA) (23 (link), 32 ). Purified DNA fragments were sent for Sanger sequencing at Eurofins Genomics (Tokyo, Japan).

To determine the relative mitochondrial DNA (mtDNA) copy number, nuclear DNA (nDNA) and mtDNA were isolated using a NucleoSpin Tissue Kit (Takara, Kyoto, Japan). The relative mtDNA copy number was calculated from the ratio of the mitochondrial gene copy number (16S rRNA gene (16S)) to nuclear gene copy number (β-actin gene (Actb)) quantified using real-time PCR (ΔΔCt analysis), as described above.

Urine was centrifuged at 4 °C for 15 min at 3,500 rpm. The urine pellet was suspended in 500 μl of cold diethylpyrocarbonate-treated PBS (pH 7.4) at 4 °C. A second 500 μl aliquot of PBS was added to wash the bottom of the 50 mL centrifuge tube to recover the remaining pellet material. The transferred pellet material in 1.0 ml of PBS was centrifuged at 12,000 rpm for 5 min at 4 °C, and the pellet was used for DNA analysis. DNA was extracted with the NucleoSpin Tissue kit (Takara, Japan) according to the manufacturer’s instructions.

Genomic DNA was extracted from BFF cells using a NucleoSpin Tissue kit and PCR amplification was performed using PrimeStar HS DNA Polymerase (Takara Bio). For reverse transcription (RT)-PCR analysis, total RNA was prepared from cells using an RNeasy Micro kit (QIAGEN) with RNase-free DNase treatment. First-strand cDNA was synthesized from 1.0 µg of total RNA using a 1st strand synthesis kit (Takara Bio) and aliquots of 1/10-1/50 of the cDNA products were used for RT-PCR analysis. The PCR primers that were used for junction PCR and RT-PCR analysis are shown in Fig. 2a and Table S1. The PCR products were directly sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 310 Genetic Analyzer (Thermo Fisher Scientific). Homologous recombination was assessed by Southern blot analysis. Two µg of genomic DNA that had been digested with ScaI or BglII were separated on 0.8% agarose gels and transferred onto nylon membranes. Digoxigenin (DIG)-labelled DNA probes (Fig. 1a) were made by using PCR DIG Labelling Mix (Roche) with the primers listed in Table S1. Hybridization and DIG detection were performed using the DIG detection system (Roche) in accordance with the manufacturer’s protocol. The CDP-Star Detection Reagent (Roche) was used to develop the membrane and the resulting chemiluminescent signal was detected using Amersham Hyperfilm ECL (GE Healthcare).