CD11c-eYFP+ cells and microglia were isolated from the brain of adult CD11c-eYFP mice, parabiotic WT/CD11c-eYFP mice or CX3CR1CreERT2-ROSA26 tdTomato mice respectively (13-16 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 SMART-Seq v4 Ultra Low Input RNA kit (#634890, Clontech) and NEBNext Ultra DNA Library Prep kit (#E7370, New England) according to the manufacturer's protocol. Briefly, 1 or 5 ng of total RNA were converted to cDNA using the SMART-seq kit. Long distance PCR was performed for 9 (5 ng) or 11 (1 ng) cycles. Agilent 2100 Bioanalyzer (High Sensitivity DNA Chip) was used for quantification and validation of the obtained cDNA. Ten ng of the resultant cDNA was fragmented using the Covaris S2 instrument in a final volume of 55 l and adjusting the settings as follows: 10% duty cycle, intensity 5, and 200 cycles per burst for 5 minutes. The resultant fragments were used for the library synthesis using the NEBNext Ultra kit. Briefly, fragments were subjected to end repair and addition of “A” bases to 3′ ends, ligation of adapters and USER excision. Library amplification was performed through 10 cycles of PCR using different indexed primers for multiplexing. All purification steps were performed using AgenCourt AMPure XP beads. Final libraries were analyzed using Agilent DNA High Sensitivity chip to estimate the quantity and to check the size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (#KK4835, KapaBiosystems). Sequencing was carried out using the Illumina HiSeq 2500, Single Reads, 50nts.
GEO Accession
GSM4696903