GSM9017904: Mouse Buffy coat, PM2.5 treated, P17; Mus musculus; RNA-Seq
RNA was extracted using TRIzol reagent (Invitrogen) and further purified with Rneasy mini plus kit (Qiagen). 1μg RNA with RIN value above 7 was used for  library preparation. RNA libraries for RNA-seq were constracted by Genewiz Inc. The First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase with random primers and Actinomycin D. The second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix (include dACG-TP/dUTP). The purified double-stranded cDNA by AxyPrep Mag PCR Clean-up (Axygen) was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of 300 bp) were recovered. The dUTP-marked second strand was digested with Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs). Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA,USA).