Truseq dna sample preparation kit

We randomly selected 6 samples of colon contents from each group for intestinal flora analysis. A TIANamp Stool DNA Kit was used to extract microbial DNA from the colon contents (DP328, TIAGEN Biotech Co., Ltd., China), following the manufacturer's instructions. The final concentration and purity of DNA were determined by using a NanoDrop (NC2000, Thermo Scientific) and 1% agarose gel electrophoresis. The V3-V4 regions of the bacterial 16S rRNA gene were amplified with a set of primers: 338F: 5-ACTCCTACGGGAGGCAGCAG-3, 806R: 5′-GGACTACHVGGGTWTCTAAT-3′, and PCR amplification was carried out in a PCR system (ABI GeneAmp® 9700, USA). The AxyPrep DNA Gel Extraction Kit (AP-GX-500, Axygen Biosciences Co., Ltd., USA) was used to purify the PCR products, and the QuantiFluor™-ST (Promega Co., USA) was used for quantification. Later, we used the TruSeqTM DNA Sample Preparation Kit (FC-121-4001, Illumina, USA) to construct the sequencing library. The microbiomes were analyzed at the Shanghai Majorbio Bio-Pharm Technology Co., Ltd., on an Illumina MiSeq platform.

Total DNA was extracted from thawed fecal samples using the QIAamp^® DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. The extracted products were determined by agarose gel electrophoresis (1% w/v agarose). Quantification of the DNA yield was carried out using a NanoDrop2000 spectrophotometer (Thermo Scientific). The DNA was stored at -20°C for Illumina MiSeq sequencing analysis.
The V3–V4 region of the 16S rRNA genes was amplified from the diluted DNA extracts with the primers 319f (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806r (5′-GGACTACH VGGGTWTCTAAT-3′). PCR amplification was then performed in a 30 μl mixture containing 0.5 μl of DMSO, 1.0 μl of forward primer (10 mM), 1.0 μl of reverse primer (10 mM), 5.0 μl of DNA sample, 7.5 μl of ddH2O and 15.0 μl of Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB). The reactions were hot-started at 98°C for 30 s, followed by 30 cycles of 98°C for 15 s, 58°C for 15 s, and 72°C for 15 s, with a final extension step at 72°C for 1 min. The PCR products were purified using an agarose gel DNA purification kit (Qiagen, Chatsworth, CA, USA). The amplicon library was prepared using a TruSeq^TM DNA sample preparation kit (Illumina Inc, San Diego, CA, USA). The sequencing reaction was conducted using Illumina MiSeq sequencing (2 × 300 bp; Hangzhou Guhe Information and Technology Co., Ltd., Zhejiang, China).

Whole genome sequences were generated using Illumina HiSeq2000 and HiSeq3000. In brief, short-insert (400-bp) libraries were constructed using the TruSeq^TM DNA sample preparation kit (Illumina, USA) for Illumina HiSeq sequencing and following the manufacturer’s protocol (Illumina). DNA from the different samples was indexed by tags and pooled together in one lane of Illumina’s Genome Analyzer for sequencing. Raw reads were first filtered to obtain the high-quality clean data by removing adaptor sequences and low-quality reads with Q-value ≤20.

Sequencing libraries were prepared using the TruSeq^TM DNA Sample Preparation kit following a standard protocol from the manufacturer (Illumina). Twelve or 24 individually indexed libraries were pooled at equal molar ratio and enriched for the X-exome using a SureSelect Human X Chromosome Exome Kit (Agilent). Each pooled library was quantified by qPCR using a KAPA library quantification kit (KAPA Biosystems) and sequenced in one lane of HiSeq2000 using 75bp pair-end sequence module.

Muscle tissue (1 mm³) was clipped from the foot of the specimen for DNA extraction. A TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) was used to extract whole genomic DNA. The mitogenome of H.aristarchorum was sequenced using an Illumina Truseq^TM DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) with paired reads measuring 150 bp in length. Quality control of raw genomic data was assessed using FastQC v.0.11.5 (Andrews 2010 ).
Quality trimming and data filtering were performed using fastp v.0.23.2 (Chen et al. 2018 (link)). Trimmed reads containing unpaired reads, more than 5% unknown nucleotides, and more than 50% bases with Q-value ≤ 20 were discarded. To evaluate the consistency of the assembly results, GetOrganelle v.1.7.7.0 (Jin et al. 2020 (link)), NovoPlasty v.4.3.1 (Dierckxsens et al. 2017 (link)) and SPAdes v.3.15.5 (Bankevich et al. 2012 (link)) were used.

WGS sequencing libraries were prepare following the previous report with slight modifications⁷¹ .
In brief, genomic DNA was extracted from snap freezing GCA tumor and matched normal tissue with DNeasy Blood & Tissue Kit (69504, QIAGEN) following manufacturer instruction. DNA concentration was measured by Qubit DNA Assay Kit in Qubit 2.0 Flurometer (Invitrogen). A total amount of 0.4 μg DNA per sample was fragmented to an average size of ~350 bp with hydrodynamic shearing system (Covaris, Massachusetts, USA) and subjected to DNA library preparation with Illumina TruSeq DNA sample preparation kit (15026486, Illumina). Sequencing was carried out on Illumina NovaSeq 6000 with 150 bp paired end mode according to the manufacturer instruction.