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 mouse tail tip fibroblasts (MTTFs) (CAST/EiJxBL6) were pelleted and stored at -80°C prior to RNA extraction. 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. Bulk RNA was extracted from cultured mouse tail tip fibroblasts using TRIzol Reagent (Invitrogen). Cell were lysed at 72°C for 10 min and placed on ice prior to first-strand synthesis. The following reverse transcriptase reaction contained 50 mM Tris-HCl (pH 8.3), 30mM NaCl, 1mM GTP, 8mM DTT, 5mM MgCl2, 2uM Smart-seq3 TSO (5'-biotin-AGAGACAGATTGCGCAATGNNNNNNNNrGrGrG-3'), 8 U Maxima H-minus RT enzyme, and 2 U recombinant inhibitor (TaKaRa) for samples containing biological inhibitor; total reaction volume 4 ul. RT thermocycles were 42°C for 90 minutes, 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 buffer, 300nM dNTP/each, 500nM MgCl2, 500nM forward primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAA*T*G-3′), 100nM reverse primer (5′-ACGAGCATCAGCAGCATAC*G*A-3′), and 0.2 U KAPA polymerase in a total reaction volume of 10 ul. Thermocycles for Smart-seq3 cDNA amplification were 98°C for 3 min, then either 20 (bulk) or 22 (single-cell) cycles of 98°C for 20 sec, 65°C for 30 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. Purified cDNA was tagmented using custom Tn5, and tagmentation reaction contained 10mM Tris-HCl pH 7.5, 5mM MgCl2, 5% dimethylformamide, and 0.08 ul (0.12ul for single cell) Tn5 and 100pg of cDNA in 2ul total, was incubated for 10 minutes at 55°C, and Tn5 was stripped from the DNA with 0.5 ul of 0.2% SDS (0.4 ul of 0.2% SDS for single-cell). To amplify libraries, a reaction mix containing the tagmented cDNA, 0.75ul of pooled dual-index primers, 1x Phusion HiFi PCR buffer, 200uM dNTP/each, and 0.1 U Phusion HiFi polymerase in 7 ul total was run was run in a thermocycler for 3 minutes at 72°C, 98°C for 3 minutes, then 12 cycles of 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, then 72°C for 5 minutes. Indexed libraries were bead purified (AMPure XP, Beckman) at a ratio of 0.6:1 beads:cDNA. MTTFs were cultured in standard medium, DMEM with 10% FBS, supplemented with 1x non-essential amino acids and 1x sodium glutamate.