Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: sequencing was performed on the Illumina NextSeq 500 instrument using the V1 chemistry high-output 150 cycles sequencing kit (Illumina). Monocyte and macrophage subpopulations were isolated via FACSorting on BD FACSAria III instrument, 0.5–1.5x105 cells were sorted into capture media (PBS, 2% BSA and 0.05% EDTA), spun down and immediately lysed in 350 ul of RLT-plus buffer (QIAGEN) supplemented with β2-mercaptoethanol. Total RNA was extracted using RNeasy Plus Mini kit (QIAGEN) and eluted in 35 ul of nuclease free water. RNA quality and quantity were assessed using Bioanalyzer 2100 instrument and RNA Pico chip. RNA-seq libraries were prepared in 96-well plate format using NEBNext Poly(A) mRNA Magnetic Isolation Module, NEBNext Ultra RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) according to the protocol. Fifty ng of total RNA were enriched for poly(A) mRNA using NEBNext Poly(A) mRNA Magnetic Isolation Module, mRNA subjected to chemical fragmentation in the presence of divalent cations at 94°C for 15 min and cDNA was generated using random primers and ProtoScript II reverse transcriptase in the presence of murine RNase inhibitor using the following program: 10 min at 25°C, 15 min at 42°C, 15 min and 70°C. Second strand DNA was generated using NEBNext Second Strand Synthesis Enzyme module (DNA Polymerase I, RNase H, E. coli DNA ligase) for 60 minutes at 16°C. Resulting double stranded DNA was purified using 1.8X volume of AMPure XP Beads (Beckman Coulter), eluted in 0.1X TE buffer, DNA ends were repaired using NEBNext End Prep Enzyme Mix (T4 PNK and T4 DNA polymerase) for 30 min at 20°C and 30 min and 65°C , followed by NEBNext adaptor for Illumina ligation using Blunt/TA Ligase master mix (T4 DNA ligase) for 15 min at 20°C and incubation with USER enzyme for 15 min at 37°C. After another round of purification using 1X volume of AMPure XP Beads, adaptor-ligated DNA was amplified using NEBNext Q5 Hot Start HiFi DNA polymerase in the presence of NEBNext multiplex oligos (dual index primers) for 14 cycles. PCR-enriched libraries were purified using 0.9X volume of AMPure XP Beads and their quality was assessed using Bioanalyzer 2100 instrument and High Sensitivity DNA chip. Equimolar amount of libraries were pooled and sequenced using an Illumina NextSeq 500.