VASA-plate / VASA-drop

VASA-seq was published in Nat Biotechnol. 2022. It is a brilliant method that can profile full-length + total RNA from single cells by adding poly-A to fragmented RNA molecules. Then the rest of the procedures are the same as a regular scRNA-seq method. The method can be done in both plate (VASA-plate) and droplet (VASA-drop) formats. The plate-based VASA-seq used the CEL-seq2 strategy for the library preparation, while the droplet-based VASA-seq used the inDrop platform combined with a picoinjection technique to precisely deliver reagents into droplets.

NOTE: Thank Jo (@Joachim De Jonghe) for providing the sequences of the VASA-drop library.


VASA-plate

Adapter and primer sequences:

Barcoded RT primer: 5'- GCCGGTAATACGACTCACTATAGGGAGTTCTACAGTCCGACGATC[6-bp UMI][6-bp cell barcode]TTTTTTTTTTTTTTTTTTTTTTTTV -3'

T7 promoter: 5'- TAATACGACTCACTATAGGG -3'

RA3 primer: 5'-/5rApp/ TGGAATTCTCGGGTGCCAAGG /3SpC3/-3'

RTP primer: 5'- GCCTTGGCACCCGAGAATTCCA -3'

Illumina RP1 primer (Truseq Small RNA kit): 5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA -3'

Illumina RPI primers: 5'- CAAGCAGAAGACGGCATACGAGAT[6-bp RPI]GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA -3'

Read 1 sequencing primer: 5'- GTTCAGAGTTCTACAGTCCGACGATC -3'

Index read primer: 5'- TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC -3'

Read 2 sequencing primer: 5'- GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Step-by-step library generation

(1) Sort single cells into plates and perform cell lysis and RNA fragmentation in the well:


5'- XXXXXXX...XXXXXX -3'    5'- XXXXXX...XXXXXX -3'    5'- XXXXXXX...XXXXXX -3'    5'- XXXXXX...XXXXXX(A)n -3'

(2) Add poly-A to fragmented RNA molecules using the E. coli Poly(A) Polymerase:


5'- XXXXXXX...XXXXXX(A)n -3'    5'- XXXXXX...XXXXXX(A)n -3'    5'- XXXXXXX...XXXXXX(A)n -3'    5'- XXXXXX...XXXXXX(A)n -3'

(3) Anneal oligo-dT24V to mRNA and reverse transcription using MMLV:


5'- XXXXXXXXXXXXXXXXXXXB(A)n
                 <-----V(T)24[6-bp cell barcode][6-bp UMI]CTAGCAGCCTGACATCTTGAGGGATATCACTCAGCATAATGGCCG -5'

(4) RNaseH and DNA Pol I based second strand synthesis:


5'- XXXXXXXXXXXXXXXXXXXB(pA)[6-bp cell barcode][6-bp UMI]GATCGTCGGACTGTAGAACTCCCTATAGTGAGTCGTATTACCGGC -3'
3'- XXXXXXXXXXXXXXXXXXXV(dT)[6-bp cell barcode][6-bp UMI]CTAGCAGCCTGACATCTTGAGGGATATCACTCAGCATAATGGCCG -5'
                                                                             ↵
                                                                     IVT starts from here

(5) Pool cells and T7 in vitro transcription to amplify cDNA (resulting in single stranded RNA):


5'- GAGUUCUACAGUCCGACGAUC[6-bp UMI][6-bp cell barcode](dU)VXXX...XXX -3'

(6) Deplete rRNA using probe-based RNaseH digestion, and add the RA3 primer for the ligation. The 3' of the RA3 primer is blocked, so only one ligation product is possible:


5'- GAGUUCUACAGUCCGACGAUC[6-bp UMI][6-bp cell barcode](dU)VXXX...XXXTGGAATTCTCGGGTGCCAAGG -3'

(7) Add the RTP primer for the reverse transcription to convert the aRNA to cDNA:


5'- GAGUUCUACAGUCCGACGAUC[6-bp UMI][6-bp cell barcode](dU)VXXX...XXXTGGAATTCTCGGGTGCCAAGG -3'
                                                       <------------ACCTTAAGAGCCCACGGTTCCG -5'

(8) Remove the RNA strand and other excessive aRNA using RNaseA, and only leave the first strand cDNA from the aRNA:


3'- CTCAAGATGTCAGGCTGCTAG[6-bp UMI][6-bp cell barcode](pA)XXX...XXXACCTTAAGAGCCCACGGTTCCG -5'

(9) Adding Illumina RP1 and PRI primers to amplify library:


5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA--------->
                                  3'- CTCAAGATGTCAGGCTGCTAG[6-bp UMI][6-bp cell barcode](pA)XXX...XXXACCTTAAGAGCCCACGGTTCCG -5'
                                                                                             <-------ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTG[6-bp RPI]TAGAGCATACGGCAGAAGACGAAC -5'

(10) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCNNNNNNNNNNNN(dT)XXX...XXXTGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAGNNNNNNNNNNNN(pA)XXX...XXXACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
             Illumina P5                    RA5             6bp   6bp        cDNA             RA3                      6bp      Illumina P7
                                                            UMI   cell                                                sample
                                                                 barcode                                              barcode

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, these are the UMI, cell barcodes and dT):


                             5'- GTTCAGAGTTCTACAGTCCGACGATC---------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAGNNNNNNNNNNNN(pA)XXX...XXXACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index sequencing primer to sequence sample index (bottom strand as template):


                                                                                5'- TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC----->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAGNNNNNNNNNNNN(pA)XXX...XXXACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, and add read 2 sequencing primer to sequence read 2 (top strand as template, these are the cDNA reads):


5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCNNNNNNNNNNNN(dT)XXX...XXXTGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                            <-------ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTG -5'

VASA-drop

Adapter and primer sequences:

Sequence used during the experiment:

*Beads-oligo-dT19V: |--5'- /5Acryd/iSpPC/CGATGACGTAATACGACTCACTATAGGGTGTCGGGTGCAG[8-bp barcode1]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG[8-bp barcode2][6-bp UMI]TTTTTTTTTTTTTTTTTTV -3'

T7 promoter: 5'- TAATACGACTCACTATAGGG -3'

Ligation adapter: 5'-/5rApp/ CTGTCTCTTATACACATCTGACGCTGCCGACGA /3SpC3/-3'

aRNA RT primer: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'

Library PCR P5 primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[8-bp i5 index]TCGTCGGCAGCGTC -3'

Library PCR P7 primer: 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp i7 index]GGGTGTCGGGTGCAG -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Read 1 sequencing primer: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'

Index 1 sequencing primer (i7): 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'

Index 2 sequencing primer (i5): 5'- CTGTCTCTTATACACATCTGACGCTGCCGACGA -3'

Read 2 sequencing primer: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'

Step-by-step library generation (the 5'-/acrydite/iSpPC/ is omitted for simplicity)

(1) Cell and beads encapsulation, and RNA fragmentation by heat:


5'- XXXXXXX...XXXXXX -3'    5'- XXXXXX...XXXXXX -3'    5'- XXXXXXX...XXXXXX -3'    5'- XXXXXX...XXXXXX(A)n -3'

(2) Add poly-A to fragmented RNA molecules using the E. coli Poly(A) Polymerase by precise picoinjection into droplets:


5'- XXXXXXX...XXXXXX(A)n -3'    5'- XXXXXX...XXXXXX(A)n -3'    5'- XXXXXXX...XXXXXX(A)n -3'    5'- XXXXXX...XXXXXX(A)n -3'

(3) Anneal oligo-dT19V to mRNA and reverse transcription using MMLV inside droplets by picoinjection of the reagents:


5'- XXXXXXXXXXXXXXXXXXXB(A)n
                 <-----V(T)19[6-bp UMI][8-bp barcode2]GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[8-bp barcode1]GACGTGGGCTGTGGGATATCACTCAGCATAATGCAGTAGC -5'--|

(4) RNaseH and DNA Pol I based second strand synthesis:


5'- XXX...XXXB(pA)[6-bp UMI][8-bp barcode2]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[8-bp barcode1]CTGCACCCGACACCCTATAGTGAGTCGTATTACGTCATCG
    XXX...XXXV(dT)[6-bp UMI][8-bp barcode2]GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[8-bp barcode1]GACGTGGGCTGTGGGATATCACTCAGCATAATGCAGTAGC -5'--|
                                                                                                        ↵
                                                                                                  IVT starts from here

(5) Break droplets and T7 in vitro transcription to amplify cDNA (resulting in single stranded RNA):


3'- XXX...XXXV(dU)[6-bp UMI][8-bp barcode2]GACAGAGAAUAUGUGUAGAGGCUCGGGUGCUCUG[8-bp barcode1]GACGUGGGCUGUG -5'

(6) Deplete rRNA using probe-based RNaseH digestion, and add the Ligation adapter for the ligation. The 3' of the adapter is blocked, so only one ligation product is possible:


3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXV(dU)[6-bp UMI][8-bp barcode2]GACAGAGAAUAUGUGUAGAGGCUCGGGUGCUCUG[8-bp barcode1]GACGUGGGCUGUG -5'

(7) Add the aRNA RT primer for the reverse transcription to convert the aRNA to cDNA:


5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG------->
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXV(dU)[6-bp UMI][8-bp barcode2]GACAGAGAAUAUGUGUAGAGGCUCGGGUGCUCUG[8-bp barcode1]GACGUGGGCUGUG -5'

(8) Remove the RNA strand and other excessive aRNA using RNaseA, and only leave the first strand cDNA from the aRNA:


5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXB(pA)[6-bp UMI][8-bp barcode2]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[8-bp barcode1]CTGCACCCGACAC -3'

(9) Adding Nextera Index primers to amplify the first strand cDNA:


5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC-------------->
                                 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXB(pA)[6-bp UMI][8-bp barcode2]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[8-bp barcode1]CTGCACCCGACAC -3'
                                                                                                                                               <--------------GACGTGGGCTGTGGG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

(10) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXB(pA)NNNNNNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNCTGCACCCGACACCCNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXV(dT)NNNNNNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNGACGTGGGCTGTGGGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
              Illumina P5          8 bp             Nextera Read 1          cDNA         6 bp   8 bp               Nextera Read 2         8 bp                   8 bp         Illumina P7
                                    i5                                                    UMI   cell                                      cell                    i7
                                                                                              barcode2                                  barcode1

Library sequencing:

(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, these are the cDNA reads, 247 cycles):


                                     5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXV(dT)NNNNNNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNGACGTGGGCTGTGGGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence cell barcode1 and i7 (bottom strand as template, 31 cycles):


                                                                                                  5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------------------------------>
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXX...XXXV(dT)NNNNNNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNGACGTGGGCTGTGGGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, and add Index 2 sequencing primer to sequence i5 (top strand as template, 8 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXB(pA)NNNNNNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNCTGCACCCGACACCCNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <-------AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5'

(4) Add read 2 sequencing primer to sequence the cell barcode2 and UMI (top strand as template, 14 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXX...XXXB(pA)NNNNNNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNCTGCACCCGACACCCNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                        <-------------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'