Easy dna kit

DNA containing exons 2–4 of PRDX1 was obtained by PCR from DT40 genomic DNA using the Easy-DNA Kit (Invitrogen, Carlsbad, California, USA) and Ex-Taq polymerase (Takara Bio Inc., Otsu, Shiga, Japan). The chicken targeting constructs for PRDX1, PRDX1-blasticidin and PRDX1-ecogpt, were generated by replacing exons 2–4 with the blasticidin or ecogpt selection marker cassette. To construct an expression plasmid carrying ggdPRDX1 cDNA (ggdPRDX1cDNA) with the tet-off promoter, ggdPRDX1 cDNA was obtained by reverse transcription-PCR (RT-PCR) using mRNA from DT40 cells using SuperScript III Reverse Transcriptase (Invitrogen) and Flag-tagged PRDX1 cDNA inserted into the pUHG 10–3 vector.

The Xoo strains used for genome sequencing in this study were collected over several decades from different rice planting areas in China (Table S1). For comparison, the represented Xoo strains belonging to 4 different races from Japan and 6 different races from Philippines were also used. The genomic DNA of each Xoo strain was extracted and purified from the overnight using the Easy-DNA kit (Invitrogen, USA), following the manufacturer’s protocol. And then the genomic DNA were sequenced by using Illumina HiSeq 2000, 2500, or 4000 to generate paired-end reads with lengths of 100, 125, or 150 bp. For each Xoo strain, raw reads were assessed with the FastQC tool (https://github.com/s-andrews/FastQC) and quality filtered using Trimmomatic (https://github.com/timflutre/trimmomatic) and bases with low quality were discarded. The filtered reads were error-corrected by library with Quake to produce clean reads.

Eighty days old homozygous (−/−) Pah^Enu2 (ENU2) and (+/+) Pah^Enu2 (WT) male mice of BTBR background strain were used for all experiments and were obtained by heterozygous mating. In a separate set of experiments, we used 3 male ENU2, 4 female ENU2 and 7 female heterozygous (+/–; HTZ) mice for the study of a possible gender effect (age: 40–60 days).
Genetic characterization was performed on DNA prepared from tail tissue using the Easy DNA kit (Invitrogen, Carlsbad, CA, USA). The ethylnitrosourea (enu2) mutation was detected after PCR amplification of exon 7 of the Pah gene and digestion thought restriction enzyme BsmAI (NewEnglandBiolabs, Inc., USA) as previously described (Pascucci et al., 2008 (link)). Mice were weaned at postnatal day (PND) 21, experimental subjects (sex matched) from different litters were housed 2–4 per cage with food and water ad libitum on a 12:12 h dark: light cycle (light on 07.00 a.m.–07.00 p.m. h).
All efforts were made to minimize the number of animals used and to alleviate their discomfort. All experimental procedures were performed in strict compliance with the Italian (D.L. 26/2014) and European Union Directive (2010/63/EU) on the protection of animals used for scientific purposes. All animal experiments were approved by the Italian Ministry of Health (Rome, Italy).
Brain tissue was collected from ENU2 and WT mice.