title: GSM4801950: AD6715; Mus musculus; RNA-Seq
library_construction_protocol: Macrophages and microglia were separated thorugh cell sorter. RNA extraction was performed using the RNeasy Micro Kit (Qiagen) RNA sequencing libraries were prepared following the SMARTseq2 protocol with some modifications. Briefly, RNA was quantified using the Qubit RNA HS Assay Kit (ThermoFisher Scientific) and the input material used for the initial cDNA synthesis varied in function of the available sample concentration (0.12ng-10ng). Reverse transcription was performed using SuperScrpit II (Invitrogen) in the presence of oligo-dT30VN, template-switching oligonucleotides (1µM) and betaine (1M). The cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kappa Biosystems), 100 nM ISPCR primer, and 15 cycles of amplification. The amplified cDNA (200 ng) was fragmented using Nextera® XT (Illumina) and amplified for 10-12 cycles with indexed Nextera® PCR primers. The Nextera® library was purified twice with Agencourt Ampure XP beads (0.8:1 ratio). The libraries were sequenced on HiSeq2500 (Illumina, Inc) in paired-end mode with a read length of 2x76bp using TruSeq SBS Kit v4 and Nextera XT Index Kit of 8bp+8bp.