title: Introducing synthetic thermostable RNase inhibitors in single-cell RNA-seq
design: Introducing synthetic thermostable RNase inhibitors in single-cell RNA-seq
library_construction_protocol: Cultured HEK293FT cells were expanded to 10 million cells, collected, filtered through a 40-μm mesh cell strainer, and resuspended in 0.5% BSA in PBS to a concentration of 2 million cells per mL for cell sorting. Cells were sorted into 96-well plates containing lysis buffer using an SH800S (Sony). Single-cell sorted plates were briefly centrifuged and kept at -80°C until they were further processed. HEK293FT cells were cultured and expanded in standard medium (DMEM/10% FBS) in 5% CO2 and 37°C. Smart-seq2 lysis buffer composition was 0.1% Triton X-100, 2.5mM dNTP/each, 2.5mM Smart-seq2 oligo-dT (5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′), and 4 U recombinant inhibitor (TaKaRa) or PVSA of the concentration specified for each condition; total buffer volume 4.5 ul. Lysis buffer with RNA was heated at 72°C for 3 min in a Bioer Life ECO thermocycler and placed on ice prior to first-strand synthesis. The following reverse transcriptase reaction contained 1x Superscript II buffer, 5mM DTT, 1M betaine, 10 mM MgCl2, 1uM Smart-seq2 TSO (5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′), 10 U Superscript II, and 10 U recombinant inhibitor (TaKaRa) for samples containing biological inhibitor; total reaction volume 10 ul. RT thermocycles were 42°C for 90 min, followed by 10 cycles of 42°C for 2 min and 50°C for 2 min. The following cDNA amplification reaction contained 1x KAPA HiFi HotStart Ready Mix and 80nM ISPCR primers (5′-AAGCAGTGGTATCAACGCAGAGT-3′); total reaction volume 25 ul. Thermocycles for Smart-seq2 cDNA amplification were 98°C for 3 min, followed by 18 (bulk), 20 (HEK), 21 (liver) or 22 (spleen) cycles of 98°C for 20 sec, 67°C for 15 sec, and 72°C for 6 min, followed by a final incubation at 72°C for 5 min. Amplified cDNA was bead purified (AMPure XP, Beckman) at a ratio of 0.8:1 beads:cDNA and inspected on a Bioanalyzer 2100 (High Sensitivity DNA kit, Agilent). Purified cDNA was tagmented using custom Tn5 (Picelli S. et al., 2014). in a tagmentation reaction which contained 2x TAPS-Mg buffer, 10% PEG 8000, 0.3nM Tn5, and 1ng of cDNA in 20ul total, incubated for 8 minutes at 55°C. Tn5 was stripped from the DNA with 3.5 ul of 0.2% SDS by incubating at room temperature for 5 minutes. To amplify tagmented libraries, a reaction mix containing the tagmented cDNA, 0.75ul of pooled dual-index primers, 1x Kapa HiFi PCR buffer, 300uM dNTP/each, and 1 U Kapa HiFi polymerase in 50 ul total was run in a thermocycler for 3 minutes at 72°C, 95°C 30 seconds, then 10 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, then 72°C for 5 minutes.