SCOPE-seq2

Preparation

A microwell array device was filled with wash buffer (20 mM Tris–HCl pH7.9, 50 mM NaCl, 0.1% Tween-20) and stored in a humid chamber one day before use.

Cell culture or tissue samples were dissociated into single cell suspension (see section, GBM tissue processing), and stained with desired fluorescent dyes.

Cell loading

The pre-filled microwell array device was flushed with Tris-buffered saline (TBS).

The single cell suspension was pipetted into the microwell array device.

After 3-min, un-trapped cells were then flushed out with TBS.

Cellular imaging

The cell-loaded microwell device was scanned using an automated fluorescence microscope (Nikon, Eclipse Ti2) under the bright-field and fluorescence channels. Bright-field images were taken using an RGB light source (Lumencor, Lida) and wide-field 10 × 0.3 NA objective (Nikon, cat# MRH00101). Fluorescence images were taken using LED light source (Lumencor, spectra x), Quad band filter set (Chroma, cat# 89402), wide-field 10 × 0.3 NA objective (Nikon, cat# MRH00101) with 470 nm (GFP channel) and 555 nm (TRITC channel) excitation for Calcein AM and Calcein red–orange, respectively.

scRNA-seq (steps performed on microwell device)

Beads (Chemgenes) were pipetted into the microwell device, and untrapped beads were flushed out with 1 × TBS. The microwell device containing the cells and the beads was connected to the computer-controlled reagent and temperature delivery system as previously described²¹.

Lysis buffer (1% 2-Mercaptoethanol (Fisher Scientific, cat# BP176-100), 99% Buffer TCL (Qiagen, cat# 1031576) and perfluorinated oil (Sigma-Aldrich, cat# F3556-25ML) was flowed into the device followed by an incubation at 50 °C for 20 min to promote cell lysis, and then at 25 °C for 90 min for mRNA capture. Wash buffer supplemented with RNase inhibitor (0.02 U/µL SUPERaseIN (Thermo Fisher Scientific, cat# AM2696) in wash buffer) was flushed through the device to unseal the microwells and remove any uncaptured mRNA molecules.

Reverse transcription mixture (1X Maxima RT buffer, 1 mM dNTPs, 1 U/µL SUPERaseIN, 2.5 µM template switch oligo, 10 U/µL Maxima H Minus reverse transcriptase (Thermo Fisher Scientific, cat# EP0752), 0.1% Tween-20) was flowed into the device followed by an incubation at 25 °C for 30 min and then at 42 °C for 90 min. Wash buffer supplemented with RNase inhibitor was flushed through the device. The device was disconnected from the automated reagent delivery system.

Exonuclease I reaction mixture (1X Exo-I buffer, 1 U/µL Exo-I (New England Biolabs, cat# M0293L)) was pipetted into the device followed by an incubation at 37 °C for 45 min. TE/TW buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.01% Tween-20) was flushed through the device.

Bead optical demultiplexing

The microwell device containing the beads with cDNAs was connected to a computer-controlled reagent delivery and scanning system (see section, “Automated reagent delivery system”).

Melting buffer (150 mM NaOH) was infused into the device and incubated for 10 min. The device was then washed with imaging buffer (2xSSC, 0.1% Tween-20). An automated imaging program scanned the device in the bright-field, Cy3 and Cy5 emission channels. Fluorescence images were acquired using an LED light source (Lumencor, spectra x), Quad band filter set (Chroma, cat# 89402), wide-field 10 × objective (Nikon, cat# MRH00101) and 555 nm and 649 nm excitation for Cy3 and Cy5, respectively. Hybridization solution (imaging buffer supplemented with probe pool A, described below) was infused into the device and incubated for 10 min. The device was then washed with imaging buffer. An automated imaging program scanned the device in the bright-field, Cy3 and Cy5 emission channels.

Repeat the previous step 7 times, with probe pool B to H.

Melting buffer was infused into the device and incubated for 10 min. The device was then washed with imaging buffer, and then disconnected from the automated reagent delivery system.

scRNA-seq (steps performed off microwell device)

Perfluorinated oil was pipetted into the device to seal the microwells. The device was then cut into 10 regions. Beads from each region were extracted separated by soaking each small piece of bead-containing PDMS in 100% ethanol, vortexing, water bath sonication, and centrifugation in a 1.7 mL microcentrifuge tube. PDMS was then removed by tweezer.

Beads extracted from each region were processed in separate reactions for the downstream library construction. Beads were washed sequentially with TE/SDS buffer (10 mM Tris–HCl, 1 mM EDTA, 0.5% SDS), TE/TW buffer, and nuclease-free water. cDNA amplification was performed in 50 µL PCR solution [1X Hifi Hot Start Ready mix (Kapa Biosystems, cat# KK2601), 1 µM SMRTpcr primer (Supplementary Table ^S6)], with 14 amplification cycles (95 °C 3 min, 4 cycles of (98 °C 20 s, 65 °C 45 s, 72 °C 3 min), 10 cycles of (98 °C 20 s, 67 °C 20 s, 72 °C 3 min), 72 °C 5 min) on a thermocycler. PCR product from each piece was pooled and purified using SPRI paramagnetic bead (Beckman, cat# A63881) with a bead-to-sample volume ratio of 0.6:1.

Purified cDNAs were then tagmented and amplified using the Nextera kit for in vitro transposition (Illumina, FC-131-1024). 0.8 ng cDNA was used as input per reaction. A unique i7 index primer was used to barcode the libraries obtained from each piece of the device. The i5 index primer was replaced by a universal P5 primer (Supplementary Table ^S6) for the selective amplification of 5′ end of cDNA (corresponding to the 3′ end of mRNA). Two rounds of SPRI paramagnetic bead-based purification with a bead-to-sample volume ratio of 0.6:1 and 1:1 were performed sequentially on the Nextera PCR product to obtain sequencing-ready libraries.

The resulting single-cell RNA-Seq libraries were pooled and 20% PhiX library (Illumina, FC-131-1024) was spiked-in before sequencing on an Illumina NextSeq 500 with a 26-cycle read 1, 58 cycle read 2, and 8 cycle index read. A custom sequencing primer (Supplementary Table ^S6) was used for read 1.