GSM5949251: P40_ctr_F1; Mus musculus; RNA-Seq
Mice were anesthetized and transcardially perfused with ice cold PBS. Brain hemispheres were homogenized and subjected to 37% / 70% percoll density gradient separation. Microglia cells were collected from the pellet of 37% percoll and washed extensively with FACS buffer (0.5% FBS in PBS) followed by staining with anti-CD11b (clone M1/70, eBioscience) and anti-CD45 (clone 30-F11, eBioscience) antibodies. Isolation of immune cells and flow cytometry. Total RNA was extracted from FACS sorted CD11b+CD45lowLin- microglia cells using The ARCTURUS® PicoPure® RNA Isolation Kit (ThermoFisher) according to manufacturer's protocol. The SMARTer v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from 500 to 750 pg 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). One-hundered and fifty pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposase. 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). Equimolar amounts of each library were pooled, and the pools were used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and the TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 or HiSeq 2000 System User Guide. Image analysis and base calling resulted in BCL files, which were converted into FATSQ files with the CASAVA1.8.2 software. Library preparation and RNAseq were performed at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany).