Long non-coding RNA sequencing (RiboZero) analysis of monocytes-differentiated macrophages. Long non-coding RNA sequencing (RiboZero) analysis of monocytes-differentiated macrophages

Analysis of lncRNA differential expression in monocytes-derived macrophages M1 and M2 at 18h, Day 3, Day 5 and Day 7. StringTie was used to perform expression level for mRNAs and lncRNAs by calculating FPKM (FPKM=[total_exon_fragments/mapped_reads(millions)×exon_length(kB)]). The differentially expressed mRNAs and lncRNAs were selected with log2 (fold change) >1 or log2 (fold change) 7.0. Approximately 1 ug of total RNA was used to remove ribosomal RNA according to the manuscript of the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA). Following purification, the ribo-minus RNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with a strand-specific library preparation by dUTP method. The average insert size for the paired-end libraries was 300±50 bp. And then we performed the pair-end 2×150bp sequencing on an Illumina Hiseq 4000 platform housed in the LC Sciences (Hangzhou, China) following the vendor's recommended protocol.