Qubit high sensitivity dna analysis

Single-cell transcriptome information was captured (from 20 sample sources) using the BD Rhapsody system. The single-cell suspension was randomly assigned to 200,000 micropores by a limited dilution method. The beads with oligonucleotide barcodes were added to the saturated state and paired with cells in the micropores. The cells were cleaved in micropores to hybridize mRNA molecules and the oligonucleotides on the beads were captured by bar code. Then reverse transcription and ExoI digestion were performed in a test tube. During cDNA synthesis, a unique molecular identifier (UMI) was bound to each cDNA molecule at the 5’ end to indicate the origin of the cell. The BD Rhapsody single-cell full transcriptome workflow includes random primer and extension (RPE), RPE amplification PCR and WTA index PCR to prepare a full transcriptome library. The high-sensitivity DNA chip (Agilent) on the bioanalyzer 2200 and the qubit high-sensitivity DNA analysis (Thermo Fisher Scientific) were used to quantify the library. Sequencing was performed by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.

Based on the 10× Genomics Chromium single‐cell platform, we performed scRNA‐seq on the PBMCs of patients.⁴⁴ Beads with unique molecular identifiers (UMIs) and cell barcodes were loaded into PBMCs in suspension in a nearly saturated state, so that each cell was paired with beads (Gel Bead‐in Emulsion). After exposure to the cell lysis buffer, the polyadenylated RNA molecules hybridized to the beads. The beads were then transferred into a single tube for reverse transcription. During cDNA synthesis, each cDNA molecule was labelled with UMI and a cell tag at the 5′ end of its source cell. To prepare a library, sequencing was used to randomly interrupt the whole transcriptome amplification product to enrich the 3′ end of the transcript connected to the cell barcode and UMI. All remaining procedures, including library construction, were conducted in accordance with standard manufacturing protocols. The sequencing library was quantified using a high‐sensitivity DNA chip (Agilent) and Qubit high‐sensitivity DNA analysis (Thermo Fisher Scientific) on bioanalyzer 2100. The library was sequenced on NovaSeq6000 (Illumina) with the 2 × 150 chemical method.

The BD Rhapsody system was used to capture transcriptome information. Quantified by a High-Sensitivity DNA chip (Agilent) on a Bioanalyzer 2200 and Qubit High-Sensitivity DNA analysis (Thermo Fisher Scientific), sequencing was performed using HiSeqXten (Illumina, San Diego, CA, USA) with a 150 bp paired-end run. scRNA-seq was performed on single-cell suspensions with viability >70%. Single-cell capture was achieved by randomly distributing a single-cell suspension in >200,000 microwells using the limiting dilution method. To pair the beads with cells, beads with oligonucleotide barcodes were added to the saturation state in the microwell. Cells were lysed in the micropores, allowing polyadenylated RNA molecules to hybridize into the beads. The beads were collected and followed by reverse transcription and ExoI digestion. During cDNA synthesis, each cDNA molecule was marked with a unique molecular identifier (UMI) at the 5’ end (that is, the 3’ end of the mRNA transcript), and a cell barcode was used to mark its cellular origin. The BD Rhapsody Single Cell Whole Transcriptome Amplification (WTA) workflow was used to prepare the entire transcriptome library, which included random primer and extension (RPE), RPE amplification polymerase chain reaction (PCR), and WTA index PCR.