Truseq dna lt sample prep kit v2

Fresh fecal contents were directly collected from the rat's cecum at the end of the study and stored in Sample Protector (TaKaRa, Dalian, China) at −80°C. The MoBio Power Fecal DNA Isolation kit (Mo BioLaboratories, Carlsbad, CA, USA) was used for DNA extraction. The quality of the extracted DNA was examined by agarose gel electrophoresis, and the OD 260/280 was analyzed by spectrophotometry. DNA libraries were prepared from 2 μg of total DNA for each sample using TruSeq DNA LT Sample Prep Kit v2 (Illumina, San Diego, California). Metagenomic sequencing was performed on HiSeq 3000 platform (Illumina, San Diego, California). After removing adapters, the raw reads were filtered to remove low-quality reads and reads that belong to the host. These high-quality reads from the samples were then assembled to contigs using Meta-Velevt. MetaGeneMark was employed to predict open reading frames (ORFs). In addition, a metagenomic catalog was generated based on the samples obtained in this study. Furthermore, the high-quality clean paired-end reads from each sample were aligned by BWA version 0.5.7-6 to the reference genes. Then the relative abundance of genes was predicted as described previously [15 (link)].

For the NGS-based sequencing, DRG tissues from 10 randomly selected pairs of CFA-treated and untreated rats were extracted. The RNA-seq library was prepared with NEBNext Ultra RNA with Poly-A selection and was sequenced on an Illumina Hi-Seq 4000. The small RNA-seq library was constructed with NEBNext Multiplex Small RNA Library Prep Kit for Illumina (#7560S). The targeted bisulfite sequencing library was prepared with a DNA library construction TruSeq DNA LT Sample Prep Kit v2 for Illumina. The C-T transition was performed using a C-T transition EpiTect Bisulfite kit from Qiagen (Germany).

Total genomic DNA was extracted from blood samples using the EasyPure® Genomic DNA Kit according to the manufacturer's instructions. Genome-wide DNA methylation profiles from four non-EMS, four CCBS-EMS, and four QSBS-EMS were examined using RRBS. Briefly, library construction was completed using the TruSeq® DNA LT Sample Prep Kit v2 (Illumina). Extracted DNA of 500 ng was digested using MspI. DNA fragments were processed by 3' and repair, A' tailing, and followed by ligation with barcoded methylated TruSeq LT adapters (Illumina). After bisulfite disposal using the Qiagen EpiTect kit, PCR amplification was performed. Multiplexed samples were pooled into a DNA library, and 100 bp paired-end sequencing was performed on HiSeq-2500 (Illumina). Quality control analysis of sequencing reads was conducted using FastQC software 7 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/available from) [22 (link)].