title: GSM3990206: SAG3; Mus musculus; RNA-Seq
library_construction_protocol: Single cell RNAseq: after eye enucleation, neural retina was removed and RPE/choroid was mechanically dissected from the sclera and digested in the presence of 1mM Hoechst to label nuclei during digestion. The cell suspension was pelleted, resuspended in sorting buffer (PBS plus 5% FBS and 2mM EDTA), filtered through a 70 µm cell strainer, stained with 1 µg/ml TO-PRO-3 and purified by fluorescence-activated cell sorting with gates set to include Hoechst+ nucleated particles and to exclude TO-PRO-3+ dead cells. Collected cells were pelleted, resuspended in sorting buffer at a concentration between 250-500 cells/µl and loaded onto the 10x Genomics Chromium platform. Bulk RNAseq of tissue-specific endothelial cells: after tissue digestion, ECs were isolated by fluorescence-activated cell sorting and RNA was extracted immediately with TRI Reagent (Molecular Research Center, Inc.) and purified with RNeasy Mini Kit (Qiagen). Bulk RNAseq of RPE/choroid tissue from Ihh+/+ and IhhiΔEC/ΔiEC mice: after eye enucleation, neural retina was removed and RPE/choroid was mechanically dissected and used for RNA extraction using the RNeasy Mini Kit (Qiagen). Bulk RNAseq of cultured choroidal GLI1+ cells: RNA was extracted with RNeasy Mini Kit (Qiagen). All cDNA libraries for bulk RNAseq assays were prepared with the TruSeq RNA Sample Preparation Kit (Illumina). Single cell RNAseq: the 10x Genomics Chromium platform was used to generate barcoded single-cell GEMs, targeting about 5000 single cells per sample. GEM-reverse transcription (53 °C for 45 min, 85 °C for 5 min) was performed in a C1000 Touch thermal cycler with 96-Deep Well Reaction Module (Bio-Rad, Hercules, CA). Next, GEMs were broken and the single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, Waltham, MA). The cDNA was amplified (98 °C for 3 min; 98 °C for 15 s, 67 °C for 20 s, 72 °C for 1 min x 12 cycles) using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Quality of the cDNA was assessed using an Agilent Bioanalyzer 2100 (Santa Clara, CA), obtaining a product of about 1170 bp. Amplified cDNA was enzymatically fragmented, end repaired, A-tailed, subjected to a double-sided size selection with SPRIselect beads (Beckman Coulter, Indianapolis, IN) and ligated to adaptors provided in the kit. A unique sample index for each library was introduced through PCR amplification using the indexes provided in the kit (98 °C for 45 s; 98 °C for 20 s, 54 °C for 30 s, and 72 °C for 20 s x 14 cycles; 72 °C for 1 min). Indexed libraries were subjected to a second double-sided size selection, and libraries were then quantified using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA). The quality was assessed on an Agilent Bioanalyzer 2100, obtaining an average library size of 455bp. Bulk RNAseq: RNA libraries were prepared for sequencing using standard Illumina protocols.
attributes.tag: GEO Accession
attributes.value: GSM3990206