title: Illumina HiSeq 3000 paired end sequencing; RNA-seq of HTNV-infected murine bone marrow-derived macrophages (mBMDM) from 0 to 36 hours
design: RNA-seq of HTNV-infected murine bone marrow-derived macrophages (mBMDM) from 0 to 36 hours
library_construction_protocol: To generate the murine bone marrow-derived macrophages (mBMDM), the femur and tibia were removed from the sacriﬁced adult mice. The bones were ﬁrst rinsed with sterile phosphate-buffered saline (PBS) containing 0.1% (v/v) penicillin-streptomycin (P/S) solution. Subsequently, the bone marrow was ﬂushed with Roswell Park Memorial Institute 1640 (RPMI 1640, Hyclone) containing 10% FBS and 0.1% P/S and filtered with the cell strainer (70 mm). Cells were resuspended with RPMI 1640 after centrifugation, and then primed with CSF (20 ng/ml, PeproTech) with medium exchange every other day for four days to generate mBMDM.  The mBMDM were infected with Hantann viruses(HTNV) at an MOI of 1 or mock infected with Co60-inactivated HTNV, and were collected for RNA-seq at 0, 12, 24 and 36 hpi (three repeats for each group). Total RNA was extracted from WT mBMDM at 0, 12, 24 or 36 hpi using the TRIzol (Invitrogen). The ribosomal RNA was removed using the Ribo-Zero™ kit (Epicentre Biotechnologies). To remove trailing sequences below a phred quality score of 20 and achieve uniform sequence lengths for downstream clustering processes, raw fastq sequences were treated with Trimmomatic tools (v 0.36) using the following options: TRAIL-ING: 20, MINLEN: 235 and CROP: 235. Sequencing read quality was inspected using the FastQC software. Adapter removal and read trimming were performed using Trimmomatic. Sequencing reads were trimmed from the end (base quality less than Q20) and filtered by length (less than 25).  Fragmented RNA (the average length was approximately 200 bp) were subjected to the first strand and second strand cDNA synthesis following by adaptor ligation and enrichment with a low-cycle according to instructions of NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB). The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit®2.0 (Life Technologies). The libraries were paired-end sequenced (PE150, Sequencing reads were 150 bp) at Guangzhou Ribo Biotechnology (Guangzhou, China) using Illumina HiSeq 3000 platform.