title: AMMA RNA-sequencing
design: Total RNA was extracted from FACS sorted CD11b+CD45lowLin- microglia cells using The ARCTURUS PicoPure RNA Isolation Kit (Thermo Fisher Scientific) according to manufacturers protocol. The SMARTer v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories) 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). 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). 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 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).