title: GSM4437381: Hyalocytes sample 7 lane 2; Homo sapiens; RNA-Seq
library_construction_protocol: Vitreous tissue samples collected in vitrectomy collection bags were immediately placed on ice in the operating theatre and processed for cell isolation within two hours of surgical resection. The content of each vitrectomy bag was centrifuged for 10 min at 250x g at 15°C. After removing the supernatant, the pellet was transferred to FACS tubes by washing with phosphate-buffered saline (PBS) buffer (PAN) and Hank's balanced salt solution (HBSS, Gibco) and again centrifuged for 10 min at 250x g at 15°C. The vitreous pellet was digested with Collagenase D (5 mg/mL, Roche) and DNase I (1 mg/mL, Roche) in HBSS for 20 min at 37°C. Cells were then filtered through a 70 µm cell strainer. Following another centrifugation step (7 min at 250x g at RT), 0.5 µL of Fixable Viability Dye (eFluor™ 780, eBioscience™) per 1 mL of cells was added. The pellet was stained for CD45 BV421 (anti-human, 1:100, BioLegend®), CD11b FITC (anti-human, 1:100, BioLegend®), CX3CR1 PE-Cy7 (anti-human, 1:200, BioLegend®). To exclude any potential contamination with blood-derived monocytes due to possible surgically induced micro bleedings which may influence the results, we further used the Anti-Human Mature Macrophages (MatMac) Antibody, an ED2-like (ectodermal dysplasia 2) marker for resident macrophages, which is absent in monocytes 8 (eFluor660, anti-human, 1:100, eBioscience™). After an incubation step of 20 min at 4°C the cells were re-suspended in FACS buffer and processed for sorting on the MoFlo Asrios EQ Cytometer (Beckman Coulter). Sorted cells were stored in RNAprotect Cell Reagent (QIAGEN) at 2-8 °C until sequencing was performed.  RNA extraction, library preparation and RNA sequencing were performed at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany; www.kfb-regensburg.de). In brief, total RNA was extracted from isolated hyalocytes and stabilized in RNAprotect Cell Reagent according to the RNeasy Plus Micro Kit protocol (QIAGEN). After pelleting, the RNAprotect buffer was removed and replaced by RLT Plus and the samples homogenized by vortexing for 30 sec. Genomic DNA contamination was eliminated 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. In total 13 samples were analyzed using RNA-sequencing. The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc.) was used to generate first strand cDNA from 750 pg total-RNA. Double-stranded cDNA was amplified by LD PCR (12 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.). 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.). Equimolar amounts of each library were pooled, and the pools then 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 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 fastq files with the bcl2fastq v2.18 software. The sequence data have been submitted to the Gene Expression Omnibus database under accession number GSE147449. The password is available from the corresponding author upon request.
attributes.tag: GEO Accession
attributes.value: GSM4437381