title: GSM4059755: EYFP 14 Sp; Mus musculus; RNA-Seq
library_construction_protocol: CD11c-eYFP+ cells and microglia were isolated from the brain of adult CD11c-eYFP mice or CX3CR1CreERT2-ROSA26 tdTomato mice respectively (13-14 weeks old) using Fluorescence Activated Cell Sorting. Brains were collected in cold HBSS buffer (w/o Ca2+ and Mg2+, Thermo Fisher Scientific #14175-053). The brain tissue was enzymatically dissociated using the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec #130-092-628). The gentleMACS™ Dissociator with Heaters (Miltenyi Biotec #130-096-427) was used for mechanical dissociation steps following the Neural Tissue Dissociation Kit (P) for dissociation without heaters. The digested tissue was filtered twice with 70 µm and 40 µm filters washing with HBSS (w/ Ca2+ and Mg2+, Thermo Fisher Scientific #14025-092). Cells ﻿were separated from myelin and debris by 30% isotonic percoll gradient (GE Healthcare #17-0891-01) in Myelin Gradient Buffer (MGB). Samples were centrifuged at 950g for 30 min without acceleration nor brake. Cells were collected from the interface, washed once with FACS Stain Buffer (BD Biosciences #554656), and processed for FACS (no staining required) in a FACSAriaII sorter (BD Biosciences) libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 (Illumina, # RS-122-2101/2) 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 (Invitrogen # 18064-014) 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 the size 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 (KapaBiosystems #KK4835) prior to amplification with Illumina's cBot. Libraries were sequenced Single Reads, 50nts on the Illumina HiSeq 2500.
attributes.tag: GEO Accession
attributes.value: GSM4059755