GSM9001996: Splenic cells isolated from mouse, old RNA, 100 G,  scFLUENT-seq; Mus musculus; RNA-Seq
Nuclei of mouse ESCs were extracted using cytolysis buffer (0.1% BSA, 200 U RNase inhibitor, 0.2% NP-40, 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2) and fixed using 1% formaldehyde. Fixed nuclei were used for biotin-tagging reaction using in-situ click working solution (50 mM Hepes pH 7.5, 2.5 mM THPTA, 2.5 mM CuSO4, 4 mM Biotin-PEG3-azide, 10 mM Sodium ascorbate) and incubated at 25°C,350 rpm with 10 s on and 10s off on a ThermoMixer for 0.5 h. The nuclei were sent to single-cell sequencing. cDNA products were amplified by PCR following 98℃ 3 min, (98℃ 10 sec, 63℃ 15 sec, 72℃ 1min) for 10 cyles, 72℃ 5 min, 4℃ Hold. The products were purified, checked for cDNA amount, and served as the template of the second PCR. Finally, after purification, one-fourth volume of cDNA was amplified by index PCR. The reaction was set on a thermal cycler with the following program: 98℃ 3 min, (98℃ 10 sec, 54℃ 15 sec, 72℃ 1min) for 7cyles, 72℃ 5 min, 4℃ Hold. The indexed sequencing libraries were purified using 0.8 DNA clean beads. Specifically, for supernatant fraction, rRNA/mtRNA blocking probes were added to the first round of PCR to remove cDNA from rRNA and mtRNA.
GEO
GSM9001996_r1