PSCSR-seq library preparation and sequencing

The schema of the PSCSR-seq method is illustrated in Fig. 1.

Cell suspensions were stained with ReadyProbes® Cell Viability Imaging Kit ({"type":"entrez-nucleotide","attrs":{"text":"R37610","term_id":"795066","term_text":"R37610"}}R37610, Thermo Fisher Scientific) for 20 min, then centrifuged (300 x g, 5 min) and resuspended in 1 x DPBS. After cell counting, the stained cell suspensions were diluted in a mix of 1 x Second Diluent (640196, TaKaRa Bio USA) and 0.4 U Ribonuclease Inhibitor (N2515, Promega) to 1 cell/35 nl. The cell suspensions were dispensed into a SMARTer ICELL8 350v Chip (640019, TaKaRa Bio USA, containing 5,184 [72 × 72] nanowells) on a MultiSample NanoDispenser (MSND, TaKaRa Bio USA). All nanowells of the ICELL8 chip were imaged with a fluorescence microscope (Olympus BX43), and the images were analyzed using CellSelect software (TaKaRa Bio USA) to determine the viability and number of cells present. Alive single cells were automatically or manually selected for the subsequent experiments.

After single-cell sorting, lysis buffer (35 nl) with 0.5% Triton™ X-100 (T9284, Sigma-Aldrich) and 4 U/μl Ribonuclease Inhibitor was dispensed into selected nanowells. The microchip was transferred to a modified SmartChip thermocycler (Bio-Rad) at 25°C for 5 min and 75°C for 10 min and chilled on ice immediately. 3'-ligation mix (35 nl) containing 0.07 pmol 3' adapter (RA3-A2N, Supplementary Table ^S3), 60 U/μl T4 RNA Ligase 2, truncated KQ (M0373S, NEB), 3x T4 RNA ligase buffer, and 1.5 U/μl Ribonuclease Inhibitor was prepared and dispensed into the previously selected nanowells. The microchip was incubated with a program of 25°C for 6 hours and 4°C for 8–10 hours, followed by 65°C for 20 min. After 3'-ligation, 35 nl of RT primer mix (0.7 pmol barcoded RT primer [SCSR-RTP, Supplementary Table ^S3], 2.5 x lambda exonuclease buffer) was added into selected nanowells, with the program “Index 1” on the MSND. The chip was placed inside a thermocycler at 70°C for 2 min and then chilled on ice. 35 nl of adapter-removed solution (2.5 U/μl Lambda exonuclease [EN0562, Thermo Fisher Scientific], 5 U/μl 5' Deadenylase [M0331, NEB], 2.5 U/μl Ribonuclease Inhibitor) was dispensed into the microchip, and incubated at 30°C for 30 min followed by 37°C for 60 min and then 75°C for 10 min. Next, 35 nl of 5'-ligation mix (0.15 pmol of the 5' adapter [SR5F, Supplementary Table ^S3], 6 mM ATP, 9 U/μl T4 RNA Ligase 1 [M0437M, NEB], 3 x T4 RNA ligase buffer, 3 U/μl Ribonuclease Inhibitor) was added into the microchip and transferred to the thermocycler with a program of 37°C for 60 min and 65°C for 20 min.

The reverse transcription reaction mix (0.9 x first-strand buffer, 50 mM DTT, 1.4 mM dNTP, 2 U/μl ribonuclease inhibitor, 28 U/μl Superscript III reverse transcriptase [18080-085, Thermo Fisher Scientific]) was dispensed into selected nanowells (35 nl of each) and incubated at 55°C for 50 min and 70°C for 15 min. 35 nl of PCR-1 mix (0.3 pmol barcoded PCR-1 primers [SR5F-P1, Supplementary Table ^S3], 0.8 mM dNTP, 3.2 x PCR buffer, and 0.16 U/μl Phanta® HS Super-Fidelity DNA Polymerase [P502-d1, Vazyme]) was dispensed into the microchip with the program “Index 2”. After dispensation, the microchip was placed in the thermocycler with a program of 95°C for 3 min, followed by 12–14 cycles (95°C for 20 s, 65°C for 20 s and 72°C for 20 s) and a final incubation at 72°C for 5 min. Finally, the microchip was inverted and centrifuged at 3000 x g for 10 min to collect and pool all contents into a single collection tube using the supplied SMARTer™ ICELL8® Collection Kit (640048, TaKaRa Bio USA).

The collected PCR-1 product was purified using 1.7 x Ampure XP beads (A63882, Beckman Coulter). The size distribution was obtained with an Agilent High Sensitivity DNA Kit (5067-4626, Agilent Technologies) on an Agilent Bioanalyzer 2100 instrument. The quantification was performed with a Qubit™ dsDNA HS Assay Kit ({"type":"entrez-protein","attrs":{"text":"Q32854","term_id":"75280861","term_text":"Q32854"}}Q32854, Thermo Fisher Scientific). The PCR-1 product was size selected with 3% agarose, dye-free, Pippin Prep (CDP3010, Sage Science) at 125–160 bp.

The 50 µl PCR-2 reaction mix was prepared with purified PCR-1 product, 0.2 mM dNTP, 1 x PCR buffer, 0.02 U/μl Phanta® Max Super-Fidelity DNA Polymerase (P505-d1, Vazyme), 0.2 µM SCSR-PCR1 primer (Supplementary Table ^S3), and 0.2 µM SCSR-PCR2 index primer (Supplementary Table ^S3). The reaction was performed with a program of 95°C for 3 min, 7–13 cycles of 95°C for 20 s, 67°C for 20 s, and 72°C for 20 s, and 1 cycle of 72°C for 5 min. The PCR-2 product was purified with 1.7 x Ampure XP beads. The PSCSR-seq library was quantified with a qPCR-based KAPA Library Quantification Kit for Illumina platforms (KK4824, Kapa Biosystems). The PSCSR-seq library was sequenced using an Illumina HiSeq2500 or NextSeq2000 instrument.