DNBSEQ-G400 paired end sequencing of SAMD00885978
Smart-seq3 libraries were prepared following the Smart-seq3 Protocol V.3. with minor modifications. Briefly, 2.4 microliter of the lysis buffer [0.4 microliter of 10x Single-cell lysis buffer (TaKaRa Bio), 0.04 microliter of 40 U/ microliter RNasin Plus Ribonuclease Inhibitor (Promega), 0.02 microliter of 100 micromolar OligodT30VN and 1.94 microliter of nuclease-free water] was added to each well of 96-well plates. Then, dissociated cells were stained with 7-AAD (Nacalai tesque), and live single cells were sorted into the plate (one cell per well) using a cell sorter (MA900, Sony Biotechnology) and stored at minus 80 degrees Celsius until lysis and reverse transcription. The cells were lysed by incubation at 72 degrees Celsius for 5 min, then kept at 4 degrees Celsius. Next, reverse transcription was performed following the SMART-seq3 library protocol V.3 with 1.6 microliter of the reverse transcription master-mix [0.1 microliter of 1 M Tris-HCl pH 8.3, 0.12 microliter of 1 M NaCl, 0.1 microliter of 100 mM MgCl2, 0.04 microliter of 100 mM GTP, 0.32 microliter of 100 mM DL-dithiothreitol, 0.05 microliter of 40 U/ microliter RNasin Plus Ribonuclease Inhibitor (Promega), 0.2 microliter of 10 mM dNTPs, 0.4 microliter of 50% Polyethylene Glycol 8000 solution, 0.08 microliter of 100 micromolar TSO (5'-biotin-AGAGACAGATTGCGCAATGNNNNNNNNrGrGrG-3'; IDT), 0.04 microliter of 200 U/ microliter Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) and 0.15 microliter of nuclease-free water] added to each well of 96-well plates. After reverse transcription and PCR pre-amplification of full-length cDNA, 100 pg cDNA was followed by Tn5 tagmentation, and library amplification of the tagmented samples was performed using custom-designed Nextera index primers containing 8-bp indexes and Tks Gflex DNA Polymerase (TaKaRa Bio).