SMARTer and Smart-seq2 on Fluidigm C1: Single-cell mESC suspension was obtained by after trypsination and passing through 30µm filter (#04-0042-2316, CellTrics). SMARTer and Smart-seq2 scripts were downloaded from Fluidigm Script Hub and run across three small C1 OpenApp IFCs (5-10µm; #100- 5759)  Wild-type E14 mouse embryonic stem cells (mESCs) were plated on 0.1% gelatin coated surfaces and cultured using Knockout DMEM (KO-DMEM, #10829, Gibco), 15% batch-tested Fetal Calf Serum (FCS, #FB-1001/500, Labtech), 1x Penicillin-Streptomycin-Glutamine (PSG, #10378-016, Gibco), 1x MEM (NEAA, #11140-035, Gibco), 2-mercaptoethanol (2-ME, #31350-010, Gibco) and 1000U Leukemia Inhibitory Factor (LIF, #ESG1107, Milipore) [1]. The mESC line (E14, #CRL-1821, ATCC) was used for BGISEQ-500 vs HiSeq4000 experiments. The mESCs were passaged every 2-3 days with daily media change.   We consider each spike-in to be detected if the estimated TPM >0. The concentration, number of molecules of each spike-in was known a priori, from the final dilution in lysis mix of scRNA-seq method. The sensitivity was calculated using a logistic regression model as previously described [1]. The detection limit was chosen as the molecular abundance where the logistic regression model passes 50% detection probability. For accuracy, we calculated the Pearson correlation between input spike-in concentration (a priori determined) and the measured scRNA-seq expression values. The accuracy for each cell was only determined if there were >7 spike-ins observed per cell. To model the relationship between sequencing depth and performance metrics (sensitivity and accuracy) for individual protocols, we use a linear model with a quadratic term for read depth to capture diminishing returns on investment We performed two biological replicates of Smart-seq2 across two different C1 IFCs and 1 SMARTer run, processed them independently.  Illumina HiSeq 4000: Single cell cDNA (1 ng/μl) from each plate-based Smart-seq2 runs of mESCs and K562 cells were independently tagmented at 55 °C for 7 minutes using in-house Tn5 transposase in a 20μl reaction volume/well . Tagmentation was stopped by adding 5μl of 0.1% SDS per well and 5 minute incubation at room temperature. PCR was performed using TruePrep Library kit (Vazyme) containing Amplify Enzyme,  Primer 1-2, N5 and N7, for Illumina sequencing. The resulting 2 libraries (mESCs and K562s) were cleaned and purified using 1x AMPure XP magnetic beads, followed by quantification of final library. Each library was sequenced across 1 lane of HiSeq V4 (Illumina) using 75bp/125bp paired-end sequencing at BGI, China.     BGISEQ-500: Single cell cDNA (1 ng/μl) from each Fluidigm based C1 IFCs (mESCs: SMARTer and 2 Smart-seq2 replicates) and plate-based Smart-seq2 (mESCs and K562s) were independently tagmented using in-house Tn5, followed by PCR amplification using using KAPA HIFI polymerase and primer set (PhoAd153 forward and reverse primer and PhoAd153 forward-tag and reverse-tag). The PCR steps were: Denaturation (95°C for 3 minutes), 15 cycles of Denaturation (98°C for 20 seconds), annealing (60°C for 15 seconds) and elongation (72°C for 25 seconds), and final elongation (72°C for 5 minutes). The libraries were cleaned, purified and size selected using 0.8x and 0.2x AMPure XP magnetic beads, and eluted in 25μl sterile nuclease-free water. The libraries were denatured in presence of splint oligo, and circularized by incubating with T4 DNA ligase (252 Units) at 37 °C four 1 hour in a 120μl final volume. Linear DNA was removed by exonuclease digestion. The libraries were then incubated with PEG32 at room temperature for 10 minutes, followed by final elution in 40μl nuclease free water and sequencing on BGISEQ-500.
Experimental Factor: library preparation
Smart-Seq2
Experimental Factor: single cell identifier
Smart-Seq2 rep2_91