Genomic dna extraction kit

Genomic DNA was extracted from whole blood with the use of commercially available QIAGEN^® genomic DNA extraction kit. High resolution HLA genotyping was performed by HistoGenetics at 4 classical major histocompatibility complex loci DRB1, DQA1, DQB1 and DPB1 using Next Generation Sequencing (NGS), as previously described [40 (link)].

A 169 kb of RP11-798L5 BAC clone (Empire Genomics., USA) containing the 150 kb human ATXN2 locus was engineered to replace the endogenous ATXN2 exon-1 CAG22 with CAG72 repeats. The BAC DNA was prepared according to published protocols [76 (link),77 ] and microinjected into FVB fertilized eggs to produce transgenic mice at the University of California Irvine (UCI) Mouse Core Facility. BAC-SCA2 mice were maintained in the FVB background and bred and maintained under standard conditions consistent with National Institutes of Health guidelines and approved by the University of Utah, IACUC protocol. For genotyping of BAC-SCA2 transgenic mice, DNA was isolated from mice tails using Qiagen genomic DNA extraction kit (Qiagen Inc., USA) and genotyping PCR was performed. Three primer sets were used to identify the transgene and the primer sequences are follows: P3 forward: 5’-AATTTATGTGATGTT CACTGTTTCTTCC-3’, P3 reverse: 5’-TACGGTCCCTCCAAATAGTGTTAC-3’, P7 forward: 5’-TCTTTTTACAGTACAAGCCCACCACC-3’, P7 reverse: 5’-TTCAAAATG CACCCTTAGCACACCTG-3’, SCA2-A forward: 5’-GGGCCCCTCACCATGTCG-3’, SCA2-B reverse: 5’-CGGGCTTGCGGACATTGG-3’. For all experiments wild-type and transgenic animals were kept as littermates. From 3 to 5 litters were used per experiment dependent on actual size of litters.

Fruit, flower, stalk, and leaf samples were harvested from a single plant of ‘Perfume Lemon’, a cultivated variety of lemon (C. limon), in Nanning, Guangxi Zhuang autonomous region, China (108.33°E, 22.84°N). Samples were frozen in liquid nitrogen and stored at −80°C until further use. Genomic DNA from young leaves and leaves with HLB symptoms were isolated using the Qiagen^® Genomic DNA Extraction Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. DNA integrity, purity, and concentration were checked using 0.75% agarose gel electrophoresis, a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA), and a Qubit Fluorometer (ThermoFisher Scientific, USA). Quantitative real-time PCR was performed on the basis of Las 16S rDNA to confirm the Las bacteria in the leaves with HLB symptoms [31 (link)]. The infected leaves were considered infected with threshold values <36 with real-time PCR (Supplementary Data Table S20) [32 (link)].
Total RNA was extracted from young fruits (YF), middle-aged fruits (MF), ripe fruits (RF), little flowers (LF), big flowers (BF), tender stalks (TS), mature stalks (MS), HLB-affected (LE_D) and healthy (LE_CK) leaves using RNAprep Pure plant Kit (Tiangen Biotech, Beijing, China). Contamination from genomic DNA was removed using RNase-Free DNase I (Takara Company, China), according to the manufacturer’s instructions.

A single 1 year old live Trachidermus fasciatus (NCBI:txid290630) fish was collected for tissue extraction. The study was conducted following Chinese law for the Protection of Animals, and the animal was treated properly and in line with the ARRIVE guidelines. Tissues from seven different areas were collected for RNA sequencing, including liver, gall bladder, stomach, heart, kidney, gill, and skin. The muscle samples were collected for both of DNA sequencing (nanopore and Illumina sequencing) and Hi-C sequencing (Illumina sequencing). The tissue samples were stored in liquid nitrogen before sequencing. Trachidermus fasciatus genomic DNA was extracted using the QIAGEN® Genomic DNA extraction kit (Cat#13323, Qiagen, Valencia, CA, USA) following manufacturer’s instructions. An RNA sequencing library was constructed using the Illumina TruSeq RNA library preparation kit. Nanopore sequencing was carried on a Nanopore GridION X5/PromethION sequencer (ONT, Oxford, UK), while Illumina sequencing (DNA, RNA, Hi-C) was performed using the Illumina HiSeq platform (Illumina, San Diego, CA, USA). Finally, the Hi-C library preparation was performed according to a previously reported protocol [17 (link)].

M.homosphaera was collected, and genomic DNA was extracted by using the QIAGEN Genomic DNA Extraction Kit (Cat# 13323, QIAGEN) according to the standard operating procedure provided by the manufacturer. The extracted DNA was detected by a NanoDrop™ One UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) for DNA purity (OD260/280 ranging from 1.8 to 2.0 and OD 260/230 is between 2.0 and 2.2), and then, a Qubit 3.0 fluorometer (Invitrogen, USA) was used to quantify the DNA accurately.