Truseq stranded rna lt guide

The library for RNA sequencing was prepared based on Illumina standard instruction (TruSeq Stranded RNA LT Guide). According to the instructions in the HiSequation 2500 user guide, the library DNA was checked for its concentration and size distribution to ensure it met the request of Illumina HiSequation 2500 system before sequencing was performed. Evaluation of reads’ quality was accomplished using FastQC [60 ]. Any reads were removed if they were less than 40 bp using Btrim (set the parameter –l = 40) [61 (link)], and remaining reads were used for further analysis. cDNA sequence file (downloaded from www.maizegdb.org) derived from Maize B73 genome assembly (V4) [52 (link)] was used as a reference. The Salmon software (version: 1.1.0) was used for reads mapping to the reference cDNA sequences and calculating the transcript per million (TPM) mapped reads of each transcript using quasi-mapping method [62 (link)]. The P-value of differential expression was calculated in the R environment (version 3.6.3, https://www.r-project.org/) using EdgeR package [63 (link)] (version: 3.28.1), for it has well performance in the identification of DEGs using three biological replicates [64 (link)]. EdgeR was downloaded from the Bioconductor website (www.bioconductor.org). GO enrichment analysis was performed using agriGO (version:2.0) [65 ].

M. marinum WT and ΔwhiB4 each were grown in 5 duplicate cultures, each containing 5 ml 7H9 media to OD600 ~1.5. 1-ml cells from 5 duplicate cultures were collected and mixed together. The bacterial cultures were pelleted and washed twice with PBS. Total RNA (1–3 µg) was isolated using the RNeasy Mini Kit (Qiagen) and purified using the RNAClean XP Kit (Beckman Coulter) and RNase-Free DNase Set (Qiagen) according to the manufacturer’s instructions. Purified RNA was used to construct cDNA library according to the TruSeq Stranded RNA LT Guide from Illumina. The concentration and size distribution of cDNA library was analyzed by Agilent 2100 Bioanalyzer and the average library size was approximately 350 bp. High-throughput sequencing was carried out on an Illumina HiSeq 2500 system according to the manufacturer’s instructions (Illumina HiSeq 2500 User Guide) and 150-bp paired-end reads were obtained. The raw reads were filtered by Seqtk and then mapped to the M. marinum M strain reference sequence (GenBank GCA_000018345.1) using Bowtie2 (version: 2–2.0.5)^{60 (link)}. Counting of reads per gene was performed using HTSeq followed by TMM (trimmed mean of M-values) normalization^{61 (link), 62 (link)}. Differentially expressed genes were defined as those with a false discovery rate <0.05 and fold-change >2 using the edgeR software^{63 (link)}. Two independent experiments were performed.

Purified RNA was used to construct cDNA library according to the TruSeq Stranded RNA LT Guide from Illumina. The concentration and size distribution of cDNA library was prepared by TruSeq Stranded Total RNA Library Prep kit and analyzed by Qubit 2.0 Fluorometer. The average library size was approximately 350 bp. High-throughput sequencing was carried out on an Illumina HiSeq 2000 system according to the manufacturer’s instructions (Illumina HiSeq 2000 User Guide) and 150-bp paired-end reads were obtained. The raw reads were filtered by Seqtk and then mapped to the M. tb H37Rv strain reference sequence (GenBank NC_018143.1) using Bowtie2 (version: 2–2.0.5) [43]. Counting of reads per gene was performed using HTSeq followed by TMM (trimmed mean of M-values) normalization [44,45]. Differentially expressed genes were defined as those with a false discovery rate <0.05 and fold-change >2 using the edgeR software [46].