Gexscope red blood cell lysis buffer

Cells were isolated from whole-blood samples, and 2 mL GEXSCOPE™ red blood cell lysis buffer (Singleron) was added at 25°C for 10 min. Single-cell suspensions with 1×10⁵ cells/mL in concentration in PBS (HyClone) were prepared. Single-cell suspensions were then loaded onto microfluidic devices and scRNA-seq libraries were constructed according to Singleron GEXSCOPE® protocol by GEXSCOPE® Single-Cell RNA Library Kit (Singleron Biotechnologies). Individual libraries were diluted to 4nM and pooled for sequencing. Pools were sequenced on Illumina HiSeq X with 150 bp paired end reads.

The fresh kidney sample was placed into the GEXSCOPE Tissue Preservation Solution (Singleron Biotechnologies) immediately at 2–8°C (15 (link)). After washed by Hanks Balanced Salt Solution (HBSS) for three times, biopsy sample was cut into small pieces (1–2 mm). Subsequently, sample was digested in 2 ml GEXSCOPE Tissue Dissociation Solution (Singleron Biotechnologies) in a 15 ml centrifuge tube by continuous agitation maintained at 37°C for 15 min. Sample was then filtered through a 40-μm sterile cell strainer (Corning). After centrifuged at 1,000 rpm for 5 min at 4°C, cell pellets were resuspended with 1 ml phosphate buffer saline (PBS) (HyClone). The cell suspension was incubated with 2 ml GEXSCOPE Red Blood Cell Lysis Buffer (Singleron Biotechnologies) for 10 min at 25°C to remove red blood cell. After centrifuged at 1,000 rpm for 5 min, the cell pellet was resuspended with PBS. Then cells were counted using a TC20 automated cell counter (Bio-Rad). Live cells were determined by trypan blue staining (Gibco). If the cell viability exceeded 85%, the subsequent sample processing was performed.

Three fresh specimens were stored in GEXSCOPE tissue preservation solution (Singleron Biotechnologies, Nanjing, China), washed three times with Hanks balanced salt solution, and minced into 1-2 mm fragments with a sterile surgical scalpel. Then, the crushed tissue was digested at 37 °C for 15 min in a 15-mL centrifuge tube containing 2 mL GEXSCOPE solution with sustained agitation. The slurry was filtered through a sterile 40 μm cell strainer and centrifuged at 1,000 rpm for 5 min. After discarding the supernatant, the sediment was resuspended in 1 mL PBS (HyClone, Logan, UT, USA). Then, each sample was treated with 2 mL GEXSCOPE red blood cell lysis buffer (Singleron Biotechnologies) for 10 min at 25 °C to remove the red blood cells. The solution was then centrifuged at 500 × g for 5 min and suspended in PBS. Finally, single cells were stained with trypan blue (Sigma, MO, USA) and microscopically evaluated for cell viability.

The fresh glioma samples were obtained during the surgeries in size of 1 cm × 1 cm × 1 cm. All samples were resected from tumor core area. Detailed sample information is listed in Supplementary Table S1. The usage of samples was approved by the Institutional Review Board at Nanjing Brain Hospital Affiliated to Nanjing Medical University. Informed consent was signed by each patient. Samples were stored in GEXSCOPE™ Tissue Preservation Solution (Singleron Biotechnologies, Nanjing, China) at 4 °C and transported to the laboratory within 6 h. The specimens were rinsed with Hanks Balanced Salt Solution (HBSS) three times and split into 1–2 mm pieces. The pieces were then digested with 2 ml of GEXSCOPE™ Tissue Dissociation Solution (Singleron Biotechnologies) at 37 °C for 15 min with sustained agitation. Then, the samples were filtered through 40 µm sterile strainers and centrifuged at 1000 rpm for 5 min, 4 °C. Afterward, the supernatants were discarded. The cell pellets were suspended in 1 ml phosphate-buffered saline (PBS; HyClone). 2 ml GEXSCOPE™ Red Blood Cell Lysis Buffer (Singleron Biotechnologies) was added to cell suspension at 25 °C for 10 min to remove the red blood cells. Subsequently, the solution was centrifuged at 1000 rpm for 5 min and suspended in PBS. Samples were counted with a TC20 automated cell counter (Bio-Rad).

Fresh tissue samples were washed with Hanks Balanced Salt Solution (HBSS) and minced into pieces. The pieces were digested in GEXSCOPE Tissue Dissociation Solution (Singleron Biotechnologies). A 40-µ sterile strainer was used to separate cells from cell debris and other impurities. The cells were centrifuged, and cell pellets were resuspended in PBS (HyClone). To remove red blood cells, GEXSCOPE Red Blood Cell Lysis Buffer (Singleron Biotechnologies) was added to the cell suspension. The mixture was then centrifuged and resuspended in PBS. Cells were counted with TC20 automated cell counter (Bio-Rad).

Fresh liver tissues were stored in GEXSCOPE^® Tissue Preservation Solution (Singleron Biotechnologies, Nanjing, China) and transported to the Singleron lab on ice as soon as possible. Specimens were washed thrice with Hank’s Balanced Salt Solution (HBSS) and minced into pieces of size 1–2 mm. Then, the tissue pieces were digested with 2 mL GEXSCOPE^® Tissue Dissociation Solution (Singleron Biotechnologies, Nanjing, China) for 15 min at 37 °C in a 15 mL centrifuge tube with sustained agitation. After digestion, 70 um sterile strainers were employed to filter samples. Next, we centrifuged samples at 50× g for 5 min at 4 °C to remove hepatocytes [24 (link),25 (link),26 (link),27 (link)]. Then, the precipitation was discarded, and the supernatant was resuspended in 1 mL of phosphate-buffered saline (PBS; HyClone, Logan, UT, USA). This action was followed by centrifuging the samples at 300× g for 5 min at 4 °C. Then, the supernatant was discarded, and the precipitation was resuspended in 1 mL of PBS. To remove red blood cells, 2 mL GEXSCOPE^® red blood cell lysis buffer (Singleron Biotechnologies, Nanjing, China) was added for 10 min at 25 °C. The solution was then centrifuged at 500× g for 5 min at 4 °C and resuspended in PBS. The samples were stained with trypan blue (Sigma, Darmstadt, Germany) and evaluated under a microscope. These cells were hepatic NPCs with few hepatocytes.