Single cell 3 chip kit

The single cell suspensions were immediately processed and loaded into a Chromium microfluidic chip of the Single Cell 3' Chip Kit (10x Genomics, CA, USA). The standard 10x Chromium Single Cell 3' protocol was followed for library preparation. Briefly, cells with specific 10x barcodes and unique molecular identifiers (UMIs) were partitioned into nanoliter-scale Gel bead-in-EMulsions (GEMs) using 10x GemCode Technology. RNA from the barcoded cells was subsequently reverse transcribed, and sequencing libraries were constructed with a Chromium Single Cell 3' reagent kit (10x Genomics). Full-length cDNA was purified using DynaBeads MyOne Silane Beads (Thermo Fischer Scientific, Waltham, USA) and amplified by polymerase chain reaction (PCR). Enzymatic fragmentation and size selection were conducted by SPRIselect Reagent (Thermo Fisher Scientific). After adaptor ligation and sample index PCR, the cDNA libraries were sequenced on an Illumina NovaSeq 6000 according to the manufacturer's instructions.

PBMCs were isolated from another blood samples of Dis6 and Con4 using the Ficoll-Paque Plus reagent (GE Healthcare). The PBMC suspensions were then loaded onto a haemocytometer for cell counting and viability examination. The viability of all the assessed samples exceeded 80%, and the cell densities of the suspensions were adjusted to 700–1200 cells/μL. The suspensions were then loaded onto microfluidic chips from the Single Cell 3′Chip Kit (10 × Genomics, CA, USA) to prepare single-cell gel beads-in-emulsions (GEMs). GEMs were subsequently subjected to cDNA library construction using a Chromium Single Cell 3′ Reagent Kit v2 (10 × Genomics). Libraries were sequenced using the BGISEQ-500 instrument. CellRanger software (version 5.0.1) was used to process the raw reads, map the reads, and quantify gene expression. Seurat software (version 3.0.2) was used for quality control and to identify highly variable genes. Single-cell RNA transcriptome analysis was then performed based on the expression matrices generated from the above steps.

Single-cell suspensions of UCB samples were loaded to chips from the Single Cell 3′ Chip Kit (10x Genomics, CA, USA) and subjected to the GemCode Single Cell Instrument (10x Genomics) to generate single-cell gel beads in emulsion, as per the manufacturer's instructions. Next, gel beads in emulsion were subjected to library construction using Chromium™ Single Cell 3′ Reagent Kits v2 (10x Genomics), the steps of which included incubation at room temperature, complementary DNA amplification, fragmentation, end repair, A-tailing, adaptor ligation, and sample index polymerase chain reaction. Because this library was designed to be sequenced by the Illumina sequencing platform, we converted the libraries to be compatible with the BGISEQ-500 sequencer. To do so, we performed a 12-cycle polymerase chain reaction on the libraries using BGISEQ adaptor primers, with subsequent DNA circularization and rolling-cycle amplification to generate DNA nanoballs. Purified DNA nanoballs were sequenced using the BGISEQ-500 sequencer, generating reads containing 16 base pairs of 10x™ barcodes, 10 base pairs of UMIs, and 100 base pairs of 3′ complementary DNA sequences. Each library was sequenced in 3 lanes, yielding ∼1.9 billion reads in total [81–83 ].