Tiangen rnaclean kit

RNA was isolated using the KANGWEI Ultrapure RNA Kit (CWBIO, China) and the isolated RNA was subjected to DNase I (Promega, Beijing, China) treatment and purified with the TIANGEN RNAclean kit (TIANGEN, China). The clean RNA samples were aliquoted into two tubes and frozed at −80°C for further RT-PCR or RNA-Seq processing. The Ribo-Zero TM Magnetic Kit (Gram-Positive Bacteria) was used to remove ribosomal RNA (rRNA) and enrich the mRNA to compensate for the low-input samples. The average RNA Integrity number (RIN) was 8, and the average RNA yield was 100 ng/μl. The library preparation, sequencing, and initial quality check were performed by Berry Genomics Corporation, Genomics and Bioinformatics Service, China (http://www.berrygenomics.com/tech-services/illumina). Samples were then sequenced using the Illumina Hiseq^TM 2500 Next Generation Sequencer at Berry Genomics Corporation, China. Three independent experiments of each group were performed and sequenced. The resulting sequences were then aligned to the reference genome of strain SK36 (GenBank Accession: NC_009009) in order to create a transcriptome map using EDGE-pro. Gene quantification was calculated by the reads per kilobase per million mapped reads method (RPKM), using RSEM (V1.2.15) software.

The experiments were performed using previous method14 (link). In brief, RHA1 was cultured in MSM to OD₆₀₀∼2.0, and then total RNA was isolated using Trizol Reagent (Invitrogen) and purified using the TIANGEN RNAclean Kit (TIANGEN) according to the manufacturer’s instructions. For qRT-PCR analysis, RNA was reverse transcribed using the M-MLV Reverse Transcriptase Kit (Promega) which was used in the qPCR reactions containing SYBR green fluorescent dye (ABI). The relative expression of mRNA was determined after normalization against 16S levels using the DD-Ct method, comparing MLDS or MLDSR expression level. qPCR was performed using an ABI StepOne PLUS PCR instrument. All primers used for qRT-PCR are shown in Supplementary Data 2. These experiments were performed in triplicate.