Ls1040 eastep super total rna extraction kit

Neuro-2a cells were transfected with pOC2ΔHOX or pcDNA plasmid for 42 h and then infected with ICP0-null virus for 5 h (MOI = 1) before being harvested for RNA extraction using a ls1040 Eastep Super total RNA extraction kit (Promega). Library preparation and sequencing were performed by Beijing Genomics Institute. A Bigseq500 platform was used to generate 150 bp paired-end reads. The raw data were filtered with SOAPnuke (v1.5.2) (https://github.com/BGI-flexlab/SOAPnuke). The clean data were mapped to the mm10+KOSnorepeat genome by Bowtie2 (version2.2.5) (https://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Gene expression levels were calculated by RSEM (version1.3.1) (http://deweylab.github.io/RSEM/). Differential expression analysis was performed using DESeq2 (version1.4.5) (https://bioconductor.org/packages/release/bioc/html/DESeq2.html).

Five samples of L. paracasei Zhang were collected at different stages of induction of the VBNC state (at 0, 3, 30, 180, and 210 days of induction), with three biological replicates for each condition. Total RNA was extracted using the LS1040 Eastep Super Total RNA Extraction Kit (Promega, Madison, WI, USA). The integrity of the extracted RNA was checked using an Agilent 2100 Bioanalyzer and agarose gel electrophoresis, and a NanoDrop2000 spectrophotometer was used to determine the purity and concentration of the extracted RNA. Qualified RNA samples (≥5 μg of total RNA amount; concentration ≥ 50 ng/μL; RNA integrity number ≥ 8.0) were used for cDNA library preparation.