Truseq pe cluster kit v3 cbot hs

The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Nova seq platform, and 150 bp paired-end reads were generated.

Two microliters of DNA (10 ng/μl) from nine fecal samples of the 0, 21, and 42 dpi groups (three samples per group) was fragmented to ~300–500 bp with a Covaris S220 (Covaris, Woburn, MA, USA) individually. Subsequently, library construction was performed using a TruSeq Nano DNA LT Sample Preparation Kit (cat. no. FC-121-4001, Illumina, San Diego, CA, USA) according to the manufacturer's instructions, and then a TruSeq PE Cluster Kit v3-cBot-HS (cat. no. PE-401-3001, Illumina, San Diego, CA, USA) was used for bridge PCR. The resulting DNA was then pooled and quantified by Kapa Library Quantification Kits (cat. no. KK4824, Kapa Biosystems, Boston, MA, USA) and sequenced using the Illumina HiSeq platform with a TruSeq SBS Kit v3-HS (cat. no. FC-401-3001, Illumina, San Diego, CA, USA).

For the preparation of RNA samples, 3 μg of total RNA from each sample served as the input for cDNA library preparation using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, E7435L, Beijing, China). The library quality was assessed based on indexing and clustering using the TruSeq PE Cluster Kit v3 cBot HS (Illumina) on the cBot system. Sequencing was conducted on the Illumina HiSeq 550. Moreover, the quality control was performed using FastQC v0.11.8, with data preprocessing by Cutadapt v1.18 to remove adapter and poly(A) sequences. Reads with more than 5% N bases were discarded via a Perl script, and those with base quality over 20 were selected with FASTX Toolkit v0.0.13. BBMap software was used for read pairing, and HISAT2 v0.7.12 aligned the high-quality reads to the mouse genome, ensuring data integrity and preparation for analysis (Deng et al. 2020 (link); Peng et al. 2019 (link)).

RNA was extracted from all 24 samples by using an NEB extraction kit following protocols by the manufacturers. RNA degradation and contamination were checked on 1% agarose gels. RNA purity was tested using a NanoPhotometer^® spectrophotometer (IMPLEN, CA, United States). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, United States). Sequencing libraries were created using the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, United States) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150-bp paired-end reads were produced.

Total RNA of each sample was extracted using TRIZOL kit according the manufacture’s protocol. RNA qualification was monitored on 1% agarose gels. The integrity and purity of total RNA were checked using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), respectively. Sequencing libraries were generated using NEBNext® Ultra™RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The Agilent Bioanalyzer 2100 system was applied to assess the library quality. The library preparations were sequenced on an Illumina platform and paired-end reads were generated, following the clustering of the index-coded sample performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Due to the relatively poor assembly of eggplant genome, the self-assembly was used in this study [61 (link)]. Transcriptome assembly was accomplished using Trinity v2.2.0 [62 (link)]. The IDs of unigenes were automatically generated by the software subsequently. The read counts of unigenes were calculated by the software RSEM v1.2.19.

RNA-seq was performed on the RNA isolated from brain tissue. A total of 3 μg RNA of each group was used for the sample preparations. The NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) was used to obtain the sequencing libraries. The TruSeq PE Cluster Kit v3-cBot-HS (Illumia) was used to cluster the index-coded samples. After cluster generation, the samples were sequenced via Illumina Hiseq platform.

A total amount of 1 μg of RNA per sample was used as the input material for RNA sample preparations. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Clustering of the index-coded samples was performed on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150-bp paired-end reads were generated. FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). We used the cluster Profiler R package to test the statistical enrichment of differentially expressed genes in KEGG pathways.