GSM5899978: Brain microglia : PLX_16; Mus musculus; RNA-Seq
RNA was extracted from samples of FACS-sorted microglia with PureLink™ RNA Micro Kit (#12183016, Invitrogen) following manufacturer indications with minor modifications. RNA was precipitated with 70% ethanol overnight at -20°C. To avoid genomic DNA contamination a DNAse step was performed. RNA quantity and purity were assessed with the High Sensitivity RNA ScreenTape® (Agilent 2200 TapeStation system). RNA libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 (#RS-122-2101/2, Illumina) according to the manufacturer's protocol. Briefly, from 2.4 to 147 ng of total RNA were used for poly(A)-mRNA selection using streptavidin-coated magnetic beads and were subsequently fragmented to approximately 300bp. cDNA was synthesized using SuperScript II reverse transcriptase (#18064-014, Invitrogen) and random primers. The second strand of the cDNA incorporated dUTP in place of dTTP. Double-stranded DNA was further used for library preparation. dsDNA was subjected to A-tailing and ligation of the barcoded Truseq adapters. All purification steps were performed with AMPure XP beads and eluted in 20 µl EB. Library amplification was performed by PCR on selected fragments using the primer cocktail supplied in the kit. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (#KK4835, KapaBiosystems) prior to amplification with Illumina's cBot. Libraries were sequenced Single Reads, 50nts on the Illumina HiSeq 2500.