Single-Cell RNA-Seq Library Preparation

Single-cell suspensions (1 × 10⁵ cells/mL) with PBS (HyClone, Logan, UT, USA) were loaded into microfluidic devices using a Singleron Matrix^® Single Cell Processing System (Singleron Biotechnologies). Subsequently, scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE^® Single Cell RNA Library Kit (Singleron Biotechnologies) [57 (link)]. Briefly, a single-cell suspension was loaded onto the microchip to partition single cells into individual wells on the chip. Cell barcoding beads were loaded into the microchip and washed. Afterwards, 100 μL of single-cell lysis buffer was added to the chip to lyse the cells and capture mRNAs at room temperature for 20 min. The beads, together with the captured RNAs, were flushed out of the microchip and used for subsequent reverse transcription, cDNA amplification, and library construction. After size selection and purification, pools were sequenced on an Illumina Novaseq 6000 (San Diego, CA, USA) with 150 bp paired-end reads.