GSM8077782: E16 10X nuc; Mus musculus; RNA-Seq
Cell dissociation and FAC-sorting: Diencephalon of the embryos were collected in ice-cold Hank's Balanced Salt Solution (HBSS) and segments were microdissected under a stereomicroscope. Thalamic chunks were then incubated in 200 µl single-cell dissociation solution consisting of papain (1 mg/ml)-enriched HBSS at 37°C for 10 min with trituration every 2 min. Cells were further dissociated via gentle up-and-down pipetting and the reaction was stopped with the addition of 400 µl of 2 mg/ml ovalbumin-enriched cold FACS buffer (2mg/mL glucose, 0.1% BSA, 1:50 Citrate Phosphate Dextrose from Sigma (C7165), 10U/mL DNase I, and 1μM MgCl2) and the cell suspension was then passed through a 40-µm cell strainer to remove cellular aggregates. Cells were then centrifuged for 5 min at 500 g at 4°C. After removal of the liquid, the pellet was suspended in 250 µl of cold FACS buffer and the resulting solution was finally sorted on a MoFloAstrios device (Beckman) to reach a concentration of 410 cells/µl. Nuclei isolation: Diencephalon of the embryos were collected in ice-cold Hank's Balanced Salt Solution (HBSS) and segments were microdissected under a stereomicroscope. Thalamic chunks were frozen and stored at -80°C until further processing. Nuclei were isolated using EZ Nuclei isolation kit as previously described. In brief, the tissue was resuspended in 2 mL of ice-cold extraction buffer (Nuclei EZ prep, Sigma NUC101), gently dounce-homogenized (KIMBLE Dounce tissue grinder, Sigma D8938, 6 strokes with A and B pestle), and incubated for 5 minutes in a total of 4 ml EZ buffer. The extracted nuclei were collected by centrifugation (500 x g at 4°C, 5 minutes) and supernatant removal followed by resuspension and incubation in EZ buffer for 5 min. After centrifugation, nuclear pellet was washed in 4 ml washing buffer containing 1% BSA in PBS, with 50U/ml of SUPERase-In (Thermo Fisher, cat# AM2696) and 50U/ml of RNasin (Promega, cat# N2611), centrifuged and nuclei were resuspended in 1 ml of washing buffer, filtered through a 30 μm strainer, stained with Hoechst (Invitrogen H3570, 1:500) for 5-10 minutes and processed for fluorescence-activated nuclei sorting on Beckman Coulter MoFlo Astrios. 20k nuclei were sorted and 42 µl of nuclei suspension was used to load the 10x Genomics snRNA-seq preparation. 10X Single cell RNA sequencing: Cell suspensions were captured using 10X Genomics Chromium Single Cell 3' v3 reagents following the manufacturer's protocol. The quality control of the cDNA and libraries was performed using Agilent's 2100 Bioanalyzer. Subsequently, the libraries were sequenced using the HiSeq 4000 sequencer. The resulting FASTQ files obtained from the sequencing were processed and mapped with 10X Genomics Cell Ranger pipeline using the GRCm38 mouse genome as a reference. Default parameters for read mapping, counting and quality controls were used as described in the Cell Ranger V6.0.0 documentation. C1 Single cell RNA sequencing: Cells stained with FlashTag were captured using the C1-Fluidigm system. Following the previously mentioned dissociation protocol, 8 µL of cell suspension was sorted on a MoFloAstrios to enrich for the top 20% most FlashTag-positive cells. Each sample representing a different embryonic time points was mixed with C1 suspension reagents (2 µL; Fluidigm) for a total cell suspension volume of approximately 10 µL, containing ~500 cells per µL. This mixture was then loaded onto the C1 Single-Cell AutoPrep integrated fluidic circuit (HT-800, Fluidigm, 100-57-80). cDNA synthesis and preamplifications were conducted following the manufacturer's instructions for the C1 system (Fluidigm). Captured cells were imaged using the ImageXpress Micro Widefield High Content Screening System (Molecular Devices). Single-cell RNA-sequencing libraries of the cDNA were prepared using the Nextera XT DNA library prep kit (Illumina). Libraries were multiplexed and sequenced in accordance with the manufacturer's recommendations, employing paired-end reads on the HiSeq4000 platform (Illumina).