Double-layer microsphere with oligonucleotide sequence for single-cell sequencing

A method for preparing a double layer microsphere with oligonucleotide sequences of claim 14, the method comprising:

• synthesizing, respectively, three oligonucleotide sequences C+D1, D2 and D3, and corresponding auxiliary sequences RC2 and RC3, wherein;
• the auxiliary sequence RC2 is a complementary sequence of ACTG [Y] ACTG, and the auxiliary sequence RC-3 is a complementary sequence of [Z] [polyT], wherein Y and Z independently represent a DNA sequence having a length of from 4 bp to14 bp, and polyT represents 10-35 T;
• adding the oligonucleotide sequence C+D1 into a first microporous plate, and subsequently adding magnetic microspheres to the first microporous plate, wherein the oligonucleotide sequence C+D1 binds to the magnetic microspheres to obtain a magnetic microsphere mixture A;
• adding a DNA ligase into the magnetic microsphere mixture A to obtain a mixed system M;
• adding the oligonucleotide sequence D2 into a second microporous plate and subsequently adding the auxiliary sequence RC2 into the second microporous plate to obtain a D2-containing microporous plate;
• adding the mixed system M into the D2-containing microporous plate, wherein the oligonucleotide sequence D1 and oligonucleotide sequence D2 bind to the magnetic microspheres to obtain a magnetic microsphere mixture B;
• adding a DNA ligase into the magnetic microsphere mixture B to obtain a mixed system N;
• adding the oligonucleotide sequence D3 into a third microporous plate and subsequently adding the auxiliary sequence RC3 into the third microporous plate to obtain a D3-containing microporous plate;
• adding the mixed system N into the D3-containing microporous plate to obtain the magnetic microspheres bound with the oligonucleotide sequences D1, D2 and D3;
• and
• coating the magnetic microspheres having the oligonucleotide sequences D1, D2 and D3 with polyacrylamide to form the plurality of double layer microspheres via a droplet microfluidic method.