title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of mouse neutrophils polarized to proinflammatory N1 by lipopolysaccharide and interferon-γ or anti-inflammatory N2 by interleukin-4 versus control
design: RNA-seq of mouse neutrophils polarized to proinflammatory N1 by lipopolysaccharide and interferon-γ or anti-inflammatory N2 by interleukin-4 versus control
library_construction_protocol: Neutrophils were isolated from mice bone marrow by Percoll gradient centrifugation. Briefly, to prevent dry-out, the bones were placed in HBSS-Prep, the ends of the bones were cut and the bone marrow (BM) was flushed into a 50 ml conical tube with HBSS-Prep. Then, the BM homogenate was centrifuged at 400xg for 5 min.  For the lysis of erythrocytes, the pellet, was resuspended in 10 ml of 0.2% NaCl (30-40 s) and then the osmolarity was restored with 10 ml 1.6% NaCl. The resulted suspension was centrifuged in 62.5% Percoll (in 1x final HBSS-Prep), for 30 min at 1000xg, brake off. At the end of centrifugation, the neutrophils-containing pellet was transferred to another 15 ml tube, wash twice with HBSS and the cells resuspended in RPMI. Centrifuge the Phasemaker™ Tubes for 30 seconds at 12,000× g prior to use. Remove growth media. Add 1mL of TRIzol™ Reagent directly to the culture dish to lyse the cells.Pellet the cells by centrifugation and discard the supernatant. Add 1 mL of TRIzol™ Reagent to the pellet. Pipet the lysate up and down several times to homogenize. Transfer the samples to the Phasemaker™ Tubes. Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex. Add 0.2 mL of chloroform per 1 mL of TRIzol™ Reagent used for lysis, then securely cap the tube. Shake vigorously by hand for 15 seconds. Incubate for 2–15 minutes. Centrifuge the sample for 5 minutes at 12,000× g at 4°C.The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upperaqueous phase. The Phasemaker™ Gel forms a barrier between the upper and lower phases. Transfer the aqueous phase (~50% of the starting TRIzol™ Reagent) containing the RNA to a new tube. Avoid puncturing or touching the Phasemaker™ Gel with the pipette when removing the aqueous phase. Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzol™ Reagent used for lysis. Incubate for 10 minutes. Centrifuge for 10 minutes at 12,000 × g at 4°C.Total RNA precipitate forms a white gel-like pellet at the bottom of the tube. Discard the supernatant with a micropipettor. Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol™ Reagent used for lysis. Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C. Discard the supernatant with a micropipettor. Vacuum or air dry the RNA pellet for 5–10 minutes.  Resuspend the pellet in 30 μL of RNase-free water by pipetting up and down. Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes. Store the RNA at –80°C. A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.