title: GSM5581286: MN_scRNA; Mus musculus; RNA-Seq
library_construction_protocol: Motor neurons cultured for 6 days were dissociated with 0.05% trypsin and stained with 0.2 uM Calcein AM and 8 uM Ethidium homodimer-1 at RT for 15 min. Single viable cells were sorted using Fluorescence Activated Cell Sorting to single wells of 96-well fully-skirted Eppendorf PCR plates in 3 uL lysis buffer (5% PEG8000, 0.1% Triton X-100, 0.5 unit/ul RNAse Inhibitor, 0.5 uM OligodT30VN, 0.5 mM dNTP in nuclease free water). The plates were immediately covered, spun at 2600 rpm for 1 min at 4°C, and stored at −80°C until library construction. Plates with cells in lysis buffer were incubated at 72 °C for 10 min for lysing the cells and denaturing the RNA. Next, 1 µl of reverse transcription mix (25 mM Tris-HCl, pH 8, 30 mM NaCl, 1 mM GTP, 2.5 mM MgCl2, 8 mM DTT, 0.5 unit/ul RNAse Inhibitor, 2 µM Smart-seq3, 2  unit/ul Maxima H-minus reverse transcriptase enzyme, was added to each well for reverse transcription and template switching at 42 °C for 90 min followed by 10 cycles of 50 °C for 2 min and 42 °C for 2 min and the reaction was inactivated at 85 °C for 5 min. cDNA pre-amplification was performed by adding 6 µl of PCR mix (1×Kapa HiFi HotStart buffer, 0.02  unit/ul KAPA HiFi DNA polymerase, 0.5 mM MgCl2, 0.3 mM dNTPs, 0.5 µM Smartseq3 forward PCR primer and 0.1 µM Smartseq3 reverse PCR primer) with program: 3 min at 98°C for initial denaturation, 19 cycles of 20 s at 98°C, 30 s at 65°C and 6 min at 72°C, followed by a final elongation for 5 min at 72°C. Pre-amplified cDNA was purified by 0.6x volume of AMpure XP beads, eluted in 14 ul nuclease free water, quantified using the Quant-iT PicoGreen dsDNA Assay Kit and size distributions were checked on a high-sensitivity DNA chip. cDNA was then diluted to 200 pg/µl, and 1 ul was used for tagmentation by mixing with 1 µl of tagmentation buffer (10 mM TAPS, pH 8.5, 5 mM MgCl2, 8% PEG8000, 0.08 ul Tn5 mix (illumina)). The reaction was performed at 55°C for 7 min, followed by heat inactivation at 80°C for 5 min. Library amplification of the tagmented samples was performed using custom-designed index primers and by adding 5 µl of PCR mix (1× Phusion High-Fidelity buffer, 0.01  unit/ul Phusion High-Fidelity DNA Polymerase, 0.2 mM dNTPs, 0.1 µM forward indexed primer and 0.1 µM reverse indexed primer). Amplification was performed as program: 3 min 72°C; 30 s at 95°C; 12 cycles of (10 s at 95°C; 30 s at 55°C; 30 s at 72°C); and 5 min at 72°C. Samples from all the wells were pooled, and then purified with Ampure XP beads at a 1:0.6 ratio. Libraries were quantified by Qubit™ dsDNA HS Assay Kit and quality checked on a high-sensitivity DNA chip. The size averaged at ~1 Kb should be expected. scRNA-seq libraries were sequenced as 100 bp paired-end reads on the Illumina NovaSeq 6000 platform and importantly, ~20% phiX spike-in was added to resolve the low complexity issue in the first 20 bps.