title: GSM3903198: T44_2.76_S420; Mus musculus; RNA-Seq
library_construction_protocol: The dissected samples in 1ml NIM buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl (pH7.4), 1mM DL-Dithiothreitol solution (Sigma 646563), 10μl of Protease inhibitor (Sigma P8340), 1.5μl of RNasin Plus RNase inhibitor (Promega, N2611), 0.1% Triton X-100, 5μM Hoechst 33342) were homogenized gently using the ice-cold dounce and pestle. After centrifuge at 3000 rpm for 8 min at 4°C, the supernatant was aspirated and gently resuspended in 1ml NIM buffer. After one repeat, the pellet was resuspended in 450μl of nuclei storage buffer (0.25M sucrose, 5mM MgCl2, 10mM Tris-HCl (pH7.4), 1mM DL-Dithiothreitol solution, 9ul of Protease inhibitor), and filtered through 40μM cell strainer on ice. The sample was incubated with 50μl of nuclease-free BSA for 1-2 hours at 4°C, then with GFP antibody conjugated with Alexa Fluor 488 (Invitrogen, A-21311) for 1 hour at 4°C. Fluorescence-activated nuclei sorting of single nuclei was performed using a BD Influx sorter with an 85μm nozzle at 22.5 PSI sheath pressure. Single cell (1 drop single) mode was used for the stringent sorting. GFP+ single nuclei were sorted into 96-well plates preloaded with 2μl of lysis buffer (Triton X-100 10%, 0.1μl RNasin Plus RNase inhibitor in H2O). After fast freeze on dry-ice, the plate was stored at -80°C before library preparation. Using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech, 634894), cDNA was amplified with 21 PCR cycle following polyA reverse transcription. The quantity and quality of cDNA was validated using the Agilent 2100 Bioanalyzer. 0.2ng/nl of amplified cDNA was used as input to prepare sequencing libraries using the Nextera XT DNA Library Preparation Kit (Illumina). Multiplexed libraries after normalized concentration were sequenced on Illumina HiSeq 4000 to generate 150bp paired-end reads.
attributes.tag: GEO Accession
attributes.value: GSM3903198