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) <-1 and with parametric F-test comparing nested linear models (p value < 0.05) by R package Ballgown. Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quality and quantity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >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.