Ultra-high-throughput single cell sequencing method

An ultra-high-throughput single cell sequencing method comprising the following steps:

• (1) preparing following reagents:
• a) molecular labeled microbeads, the molecular labeled microbead comprising a microbead body and a coupled molecular label sequence, the molecular label sequence comprising in sequence:
• a universal primer sequence, serving as a primer binding region during PCR amplification;
• a first cell barcode sequence;
• and
• a first bridge sequence;
• b) a reverse transcription sequence for intracellular reverse transcription, the reverse transcription sequence comprising in sequence:
• a second bridge sequence;
• a second cell barcode sequence, forming a cell barcode sequence in conjunction with the first cell barcode sequence, the cell barcode sequence being used for identifying a cell from which an mRNA to which each sequence in a constructed sequencing library corresponds is derived;
• a molecular barcode sequence for identifying mRNA to which each sequence in a constructed sequencing library corresponds;
• a poly-T tail for complementary pairing with intracellular mRNA with a poly-A sequence;
• and
• c) a bridge primer for ligating the above-mentioned label sequence in a) and the reverse transcription sequence in b), the bridge primer having, on both ends, a sequence complementary to the first bridge sequence and the second bridge sequence, respectively;
• (2) adding the reverse transcription sequence to a cell sample to be sequenced for intracellular reverse transcription such that the poly-T tail of the reverse transcription sequence is ligated with a cDNA sequence derived from reverse transcription of the intracellular mRNA sequence to obtain a reverse transcription sequence-cDNA sequence;
• (3) compartmentalizing one or more of the intracellularly reverse-transcribed cells from step (2) with one molecular labeled microbead by microwell-plate technology or microfluidic technology, and lysing the cells under the action of a lysis buffer, incubating, and then ligating the first bridge sequence with the second bridge sequence by the pairing of bridge primers with the first bridge sequence and the second bridge sequence, respectively, followed by ligation through a ligase, such that a molecular label sequence-reverse transcription sequence-cDNA sequence coupled to the microbead is obtained;
• (4) collecting the microbeads coupled with the molecular label sequence-reverse transcription sequence-cDNA sequence, and performing PCR amplification to obtain a cDNA sequence having the first cell barcode sequence, the second cell barcode sequence and the molecular barcode sequence;
• and
• (5) constructing a cDNA sequencing library with the product obtained from step (4), and then performing high-throughput sequencing to obtain information of specific transcriptome of millions of single cells;
• wherein, the preparation method of the molecular labeled microbeads comprises the following steps:
• classifying the bridge primers for synthesizing the molecular label sequences into a plurality of primers in accordance with a number of specific fragments, each primer comprising a specific fragment, there being adapter sequences between each of the primers for bridging, ligation and complementing each other, wherein a primer corresponding to a 5′ end of the molecular label sequence further comprises the universal primer sequence, and a primer corresponding to a 3′ end of the molecular label sequence further comprises the first bridge sequence;
• and
• coupling the primer corresponding to the 5′ end of the molecular label sequence to the microbead body, then annealing and extending remaining primers in sequence by PCR, and cascading remaining specific fragments of the molecular label sequence in sequence from the 5′ end to the 3′ end to prepare and obtain the molecular labeled microbead;
• and
• wherein, the molecular label sequence is:
• 5′-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAACGCAGAGTACGTNNNNNN CGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNTCGTCGGCAG CGTC-3′ (SEQ ID No. 4), wherein, N represents any one of A/T/C/G and is randomly synthesized.