title: GSM2879332: 09_CNS_microglia; Mus musculus; RNA-Seq
library_construction_protocol: Mice were sacrificed and spinal cords were isolated and transferred to ice-cold PBS (Sigma-Aldrich). Tissue was then cut in small pieces and digested in RPMI containing 2% fetal calf serum (Sigma-Aldrich), 25mM HEPES (Sigma-Aldrich), DNase I (10ng/ml, StemCell Technologies) and Collagenase D (0,8mg/ml, Roche) for 30 min at 37°C. Reaction was stopped by addition 1:100 dilution of a 0.5M EDTA (Sigma-Aldrich) solution. Suspension was filtered through 70 µm cell strainers (Falcon) and resuspended in a 30% solution of Percoll (Sigma-Aldrich). After 30 min of gradient centrifugation at 10.800 r.p.m., the top (myelin) and lower (red cells) layers were removed and the remaining solution was filtered through 70µm cell strainers (Falcon) before further analysis. Cells were stored and shipped in RNAprotect buffer at 2-8 °C. After pelleting, the RNAprotect buffer was replaced by RLT Plus and the samples were homogenized by vortexing for 30 sec. Genomic DNA contamination was removed using gDNA Eliminator spin columns. Next, ethanol was added and the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Finally, total RNA was eluted in 12 μl of nuclease-free water. Purity and integrity of the RNA were assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico LabChip reagent set (Agilent, Palo Alto, CA, USA). The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from 1 ng total-RNA. Double stranded cDNA was amplified by LD PCR (11 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step, 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA).
attributes.tag: GEO Accession
attributes.value: GSM2879332