GSM5849526: Primary microglia-mock rep2; Mus musculus; RNA-Seq
GSM5849526
The RNA of all cells was harvested using Trizol reagent. A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using Hieff NGS™ MaxUp Dual-mode mRNA Library Prep Kit for Illumina®(12301ES96, YEASEN, China) 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 1nd Strand Synthesis Reaction Buffer First. Second strand cDNA synthesis was subsequently performed using 2nd Strand Synthesis Buffer. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, adaptor was ligated to prepare for library. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with Hieff NGS™ DNA Selection Beads DNA(12601ES56, YEASEN, China). Then PCR was performed with 2×Super CanaceTM High-Fidelity Mix，Primer mix and Adapter Ligated DNA. At last, PCR products were purified (Hieff NGS™ DNA Selection Beads) and library quality was assessed on the Qubit®2.0 Flurometer. The libraries were then quantified and pooled. Paired-end sequencing of the library was performed on the MGISEQ2000 sequencers .