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. Cells were sorted into 384 well plates each containing 3 μl Vapor-Lock (Qiagen) in all wells and eight different conditions of 0.3 µl lysis buffer consisting of 0.125µM OligodT30VN (5'-Biotin-ACGAGCATCAGCAGCATACGAT30VN-3' ; IDT) adjusted to reverse transcription (RT) volume, 0.5mM dNTPs/each adjusted to RT volume, 0.1% Triton X-100, 5% PEG8000 adjusted to RT volume, and the indicated type and amount of RNase Inhibitor (no RNAse inhibitor, 0.003 µl RNase Inhibitor (40 U/μl, Cat. 2313B, TaKaRa), SEQURNA Thermostable RNase inhibitor (Cat. SQ00201, SEQURNA); 0.06 U/µl, 0.15 U/µl, 0.3 U/µl, 0.6 U/μl, 1.5 U/µl, 3.0 U/µl). After cell sorting plates were briefly centrifuged before put to -80°C immediately after sorting (day 0). To test the effect of temperature and time on single cell RNA stability for each of the eight lysis conditions, 384 well plates containing sorted cells in lysis buffer were left at either room temperature (25 °C) for 1, 4, or 7 days or in fridge (4°C) for 1, 4, 7, or 14 days, before commencing library preparation. To serve as control (day 0), two plates were extracted immediately from -80°C immediately before library preparation.  Before RT, plates were denatured at 72°C for 10 min followed by addition of 0.1 µl of RT mix; 25mM Tris-HCL pH 8.4 (Fischer Scientific), 30mM NaCl (Ambion), 1mM GTP (Thermo Fisher Scientific), 2.5mM MgCl2 (Ambion), 8mM DTT (Thermo Fisher Scientific), 0.75uM Template Switching Oligo (TSO) (5′-Biotin-AGAGACAGATTGCGCAATGNNNNNNNNWWrGrGrG-3′; IDT), 0.25 U/ µl RRI (Cat. 2313B, TaKaRa), and 2 U/µl of Maxima H Minus reverse transcriptase (Cat. EP0752, Thermo Fisher Scientific). RRI was excluded from the RT mix (replaced with water) in case of SEQURNA samples. Plates were quickly centrifuged after dispensing to ensure merge of lysis and RT volumes underneath the Vapor-lock overlay and incubated at 42 °C for 90 min, followed by ten cycles of 50 °C for 2 min and 42 °C for 2 min. After RT, 0.6 µl PCR mix was dispensed to each well containing the following: 1× SeqAmp PCR buffer (Takara Bio), 0.025 U µl−1 of SeqAmp polymerase (Takara Bio) and 0.5 µM Smartseq3 forward (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG-3′; IDT) and reverse primer (5′-ACGAGCATCAGCAGCATACGA-3′; IDT). Plates were quickly spun down before being incubated as follows: 1 minute at 95 °C for initial denaturation, 12 cycles of 10 seconds at 98 °C, 30 seconds at 65 °C and 4 minutes at 68 °C. Final elongation was performed for 10 minutes at 72 °C. After PCR, pre-amplified libraries were diluted with 9 µl H2O, before transferring 1 µl of diluted cDNA from each well into a new 384 well plate. Tagmentation was performed by adding 1 µl of tagmentation mix; 1x tagmentation buffer (10 mM Tris pH 7.5, 5 mM MgCl2, 5% DMF), 0.003 µl Tagmentation DNA Enzyme 1 (TDE1; Illumina DNA sample preparation kit) to the 1µl of diluted cDNA per well. At this step the tagmentation mix contains SEQURNA RNase inhibitor at a concentration of 0.015x what was in the lysis buffer. Plates were incubated for 10min at 55°C before the reaction was stopped by the addition of 0.5 µl 0.2% SDS to each well. Index PCR was carried out after the addition of 3.5µl custom Nextera Index primers (0.5µM) by dispensing 2 µl of PCR mix; 1× Phusion Buffer (Thermo Fisher Scientific), 0.01 U µl−1 of Phusion DNA polymerase (Thermo Fisher Scientific), 0.025% Tween-20, 0.2 mM dNTP each. PCR was performed out at 3 minutes at 72 °C; 30 seconds at 95 °C; 12 cycles of (10 seconds at 95 °C; 30 seconds at 55 °C; 1 minute at 72 °C); and 5 minutes at 72 °C. Each indexed library plate was pooled by spinning out gently using a 300-ml robotic reservoir (Nalgene) fitted with a custom scaffold by pulsing the centrifuge to < 200g. The pooled libraries were afterwards purified with custom carboxylated magnetic beads in 22% PEG solution at a ratio of 1 sample to 0.7 beads.