GSM5775670: Retinal microglia P6 FC1; Homo sapiens; RNA-Seq
Total RNA was extracted from FACS sorted cells stabilized in RNAprotect buffer ac-cording to the “Purification of total RNA from animal and human cells” protocol of the RNeasy Plus Micro Kit (QIAGEN, Hilden, Germany). In brief, cells were stored and shipped in RNAprotect buffer at 2-8 °C. After pelleting by centrifugation for 5 minutes at 5,000 x g, RNAprotect was replaced by 350 µl RLT Plus buffer and the samples were homogenized by vortexing for 30 seconds. Genomic DNA contamina-tion was removed by using gDNA Eliminator spin columns. Next, one volume of 70 % ethanol was added and the samples were applied to RNeasy MinElute spin col-umns followed by several wash steps. Finally, the 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). RNA isolation from FFPE retinal specimens was carried out as previously described (Lange et al., 2018;Schlecht et al., 2020a). Briefly, total RNA was extract-ed from FFPE samples using the Quick-RNA FFPE Kit (Zymo Research, Irvine, Cali-fornia). Following DNAse I digestion using the Baseline-ZERO Kit (Epicentre, Madi-son, Wisconsin), the RNA concentration was quantified using the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, Carlsbad, California). RNA quality was determined via the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, Waltham, Massachusetts). 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 approx-imately 1 ng total-RNA. Double-stranded cDNA was amplified by LD PCR (12 cy-cles) 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 frag-mented) 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 Library Quantification Kit - Illumina/ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA). Equimolar amounts of each library were sequenced on a NextSeq 500 instrument controlled by the NextSeq Control Software (NCS) v2.2.0, using two 75 Cycles High Output Kits with the dual index, single-read (SR) run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA) v2.4.11. The resulting .bcl files were converted into .fastq files with the bcl2fastq v2.18 software. RNA extraction, library preparation and RNAseq were performed at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany; www.kfb-regensburg.de). Retinal samples (FFPE) were sequenced using the 3' RNA sequencing method Massive Analysis of cDNA Ends (MACE) (Schlecht et al., 2020a;Wolf et al., 2020), which allows se-quencing of FFPE samples with high accuracy and without a significant impact of FFPE processing (Boneva et al., 2020a). Briefly, barcoded libraries comprising unique molecule identifiers were sequenced on the NextSeq 500 (Illumina) with 1 × 75 bp. PCR bias was removed using unique molecular identifiers.