Picopipet

Human lung cancer cell lines used in this study were from the established collections in our labs or as reported in our previous studies [8 (link)–10 (link)]. PC-9 erlotinib resistant cells were established from PC-9 cells by stepwise exposure to erlotinib from 0.005 μM to 5 μM for about 4 months, and the clone named PC-9 ER clone 5 was isolated with PicoPipet (Nepa Gene, Chiba, Japan). Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1x penicillin/streptomycin solution (Mediatech, Inc., Manassas, VA) at 37°C in a humidified tissue culture incubator with 5% CO₂. All experiments using acquired resistance cells were performed following the removal of drug exposure to avoid the direct effects of drugs on PD-L1 expression. IFN-gamma (Cell Signaling Technology, Dancers, MA) stimulation for 24 hours was performed to mimic an immune cell interaction.

Oocyst samples were purified in several steps before DNA preparation. For each DNA preparation, approximately 500 morphologically similar oocysts were collected by using PicoPipet (Nepa Gene, Japan). Then the oocysts were washed three times with distilled water and finally concentrated to a volume of 50 μL. Before DNA extraction, the oocyst samples were transferred to a 1.5 mL centrifugal tube and five cycles of freezing/thawing were performed; quick freezing was performed in a −80 °C freezer for 5 min, while quick thawing was conducted in a 37 °C water bath for 5 min. Oocysts were crushed using 0.2 mm glass beads (Biomedical Science, Tokyo, Japan) followed by vortexing for 5 min at 2000 rpm [13 (link)]. Thereafter, DNA was extracted from the lysate using a PowerFecal^® DNA isolation kit (MO BIO Laboratories, USA), according to the manufacturer’s instructions. The DNA was quantified spectrophotometrically and stored at −20 °C for subsequent analysis.

Prior to micromanipulation-based single-cell isolation, a spacer seal (a slide seal for in situ PCR, inner space: 9 × 9 mm², thickness: 300 μm, Thermo Fisher Scientific, Waltham, MA, USA) was affixed at the glass slide (Matsunami Glass, Osaka, Japan). Cellular RNA was stabilized using CellCover (AL Anacyte Laboratories UG, Hamburg, Germany), if required, according to the manufacturer’s protocol. After determination of cell concentration by a hemocytometer, ~100 cancer cells were suspended in 100 μl of PBS-EB. The cell suspension was mounted only on the inside of the spacer seal and observed under a fluorescence microscope (IX71; Olympus Co., Tokyo, Japan). Single cancer cells exhibiting strong florescence were manually picked up by a micromanipulator equipped with a 30 μm diameter glass capillary (PicoPipet; Nepa Gene Co., Ltd., Chiba, Japan). Then, single cells were transferred into 0.4 μL of a lysis buffer (0.5% NP40) in 200 μL PCR tubes (N8010840, Thermo Fisher Scientific). In this study, we use 0.5% NP40 as lysis buffer for subsequent Quartz-seq based whole transcriptome amplification. Isolated single-cells were immediately frozen with liquid nitrogen and stored at −80 °C for further whole transcriptome amplification.