title: GSM8484777: Macrophages, IL4 stimulation + Ti64_CaP_Zn25, donor 1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using the E.Z.N.A. total RNA kit I (Omega Bio-tek, Norcross, USA) according to the manufacturer's instructions. 500ng of total RNA was used for construction of sequencing libraries. The quality of total RNA samples was checked on BioAnalyzer2100 (G2939BA, Agilent, USA) using Eukaryotic Total RNA Nano Kit (5067-1512, Agilent, USA) according to manufacturer's protocol. Samples with RNA integrity number (RIN) value >8 were accepted for the library preparation process. RNA-Seq libraries were prepared from Illumina Stranded mRNA Prep and Ligation Kit (20040534, Illumina, USA) according to the manufacturer's protocol. Briefly, Oligo(dT) magnetic beads purify and capture the mRNA molecules containing polyA tails. Then the purified mRNA was fragmented and copied into first strand complimentary DNA (cDNA) using reverse transcriptase and random primers. In the second strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity. The final steps add adenine (A) and thymine (T) bases to fragment ends and ligate adapters. Sequencing runs are performed on an Illumina NextSeq 550 instrument using single-end 75-cycle sequencing by the NextSeq 500/550 High Output Kit v2.5 (75 Cycles) (20024906, Illumina, USA).