title: GSM8652525: J774A.1 cells, M2_1; Mus musculus; RNA-Seq
library_construction_protocol: Cell lysis and homogenization: Collect the cell suspension and add three volumes of TRIzol reagent (for tissue samples, add 1.5 mL TRIzol); Phase separation: Add 1/5 volume of chloroform to the total volume. Cap the tube tightly, vortex for 15 seconds, and incubate at room temperature for 2-3 minutes; Centrifugation: Centrifuge at 2,000 rpm for 10-15 minutes at 4°C; RNA precipitation: Carefully transfer the upper aqueous phase to a new tube and add an equal volume of isopropanol. Invert to mix and incubate at room temperature for 10 minutes; Pelleting RNA: Centrifuge at 12,000 rpm for 10 minutes at room temperature or 4°C; Washing: Carefully remove the supernatant and add 1 mL of 75% ethanol prepared with DEPC-treated water. Wash the tube cap and wall thoroughly and gently flick the tube to resuspend the pellet. For each mL of Total RNA Extraction Reagent used, wash the pellet with 1 mL of 75% ethanol; Final centrifugation: Centrifuge at 12,000 rpm for 3 minutes at room temperature or 4°C. Carefully remove the supernatant, taking care not to lose the RNA pellet; Drying and resuspension: Air-dry the pellet for 2-3 minutes. Resuspend the pellet in 30-100 μL of RNase-free water (DEPC-treated water) and dissolve completely. Measure a small aliquot for quality control, and store the remaining 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 250~300 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, adaptor-ligated 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.