Truseq standard mrna library prep kit

Worms were harvested, washed three times with M9 medium, resuspended in 1 ml of TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) and frozen in liquid nitrogen. Worms were broken open by five repeats of freeze and thaw using liquid nitrogen and a 42°C heating block, before RNA was extracted and purified according to the supplier’s protocol with the modification that RNA was incubated with 50% 2-propanol at -80°C overnight for efficient precipitation. The purified RNA was treated with DNA-free Kit (Thermo Fischer Scientific, Waltham, MA, USA) to remove DNA. For mRNA quantification by RT-qPCR, cDNA was generated from total RNA by ImProm-II Reverse Transcription System (Promega, Fitchburg, WI, USA) using oligo(dT)₁₅ primers (for mature mRNAs) or random primers (for pre-mRNAs) according to the supplier’s protocol. RT-qPCR was performed with specific primers (S4 Table), the SYBR Green PCR Master Mix (Applied Biosystems), and the StepOnePlus Real-time PCR System. After 40 cycles of PCR amplification, some samples were subjected to agarose gel electrophoresis (Fig 6F). For poly(A)-RNA sequencing, libraries were prepared using the TruSeq Standard mRNA Library Prep Kit (Illumina, San Diego, CA, USA) and sequenced.

Total RNA was purified from BMDMs using TRIzol reagent (Invitrogen) and the cDNA library was prepared with the TruSeq Standard mRNA Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. The libraries were sequenced using a NovaSeq 6000 platform (Illumina) by Macrogen. Qualified reads were mapped to the Mus musculus mm10 reference genome by HISTA2 version 2.1.0. and assembled using StringTie version 2.1.3b. Differentially expressed genes (DEGs) were identified with DESeq2, and a more than 1.5-fold change with a p value less than 0.05 was considered statistically significant. Functional annotation of DEGs was performed with DAVID (https://david.ncifcrf.gov), and the statistically enriched pathway was analyzed using the KEGG database in DAVID. A heat map of the relative expression pattern of the DEGs was created with the heatmap.2 function of the gplot package in RStudio version 3.4.4.