FIPRESCI

FIPRESCI borrows the idea from scifi-RNA-seq, but it is designed to work on the 10X Genomics 5' system. It uses barcoded Tn5 to add the first round of index to the DNA/RNA hybrid after reverse transcription. Then cells are overloaded into the 10X system. In this way, it offers an ultra-high throughput way of profiling the 5' of RNAs in single cells. The full oligo sequences details are taken from the Supplementary Table 1 from their Genome Biology publication.

Adapter and primer sequences:

Beads-TSO (PN-220112): |--5'- CTACACGACGCTCTTCCGATCT[16-bp GEM barcode][10-bp UMI]TTTCTTATATrGrGrG -3'

RT PolyT Primer (NEB S1327S): 5'- TTTTTTTTTTTTTTTTTTTTTTTVN -3'

RT random Primer (Thermo Scientific SO142): 5'- NNNNNN -3'

TN5_A_ME: 5'-/Phos/ CTGTCTCTTATACACATCT /3ddC/-3'

TN5_R2_index: 5'- GACGTGTGCTCTTCCGATCT[6-bp Tn5 barcode]AGATGTGTATAAGAGACAG -3'

S5R-P5-bio: 5'-/Bio/ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC -3'

S5R-P5: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC -3'

S-P7-index: 5'- CAAGCAGAAGACGGCATACGAGAT[6-bp i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3'

Illumina TruSeq Read 1 primer: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'

Illumina TruSeq Read 2 primer: 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3'

Sample index sequencing primer: 5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Step-by-step library generation

(1) Reverse transcription with Poly-dT and random RT primer (omitted afterwards) using MMLV:


    5'- XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXB(A)₂₃
                   <--------NNNNNNN        <-------V(T)₂₃ -5'

(2) The terminal transferase activity of MMLV adds extra Cs:


    5'- XXXXXXXXXXXXXXXXXXXXXXB(A)₂₃
     CCCXXXXXXXXXXXXXXXXXXXXXNV(T)₂₃ -5'

(3) Anneal "TN5_A_ME+TN5_R2_index" and use those to assemble the barcoded Tn5 transposomes (this is a Tn5 homo-dimer). Then use the assembled transposome to tag the RNA/DNA hybrid (will create 9-bp gaps):


Product 1 (middle of the transcript, cannot be captured by the 10X system, omitted afterwards):

5'- GACGTGTGCTCTTCCGATCT[6-bp Tn5 barcode]AGATGTGTATAAGAGACAGXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                          TCTACACATATTCTCTGTC      XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA[6-bp Tn5 barcode]TCTAGCCTTCTCGTGTGCAG -5'


Product 2 (3' of the transcript, cannot be captured by the 10X system, omitted afterwards):

5'- GACGTGTGCTCTTCCGATCT[6-bp Tn5 barcode]AGATGTGTATAAGAGACAGXXXXXXXXX...XXXB(pA) -3'
                                          TCTACACATATTCTCTGTC      XXX...XXXV(dT) -5'


Product 3 (5' of the transcript, can captured by the 10X system in the next step):

5'-   XXX...XXX         CTGTCTCTTATACACATCT
   CCCXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA[6-bp Tn5 barcode]TCTAGCCTTCTCGTGTGCAG -5'

(4) Pool all cells and overload to the 10X Genomics 5' system to captured cDNA by gel bead with barcoded TSO:


|--5'- CTACACGACGCTCTTCCGATCT[16-bp GEM barcode][10-bp UMI]TTTCTTATATGGGXXX...XXX         CTGTCTCTTATACACATCT
                                                         <-----------CCCXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA[6-bp Tn5 barcode]TCTAGCCTTCTCGTGTGCAG -5'

(5) cDNA cleanup and add S5R-P5-bio primer to enrich cDNA (single-primer linear PCR):


5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-------->
                                 |--5'- CTACACGACGCTCTTCCGATCT[16-bp GEM barcode][10-bp UMI]TTTCTTATATGGGXXXXXXXXX...XXXXXXXXXCTGTCTCTTATACACATCT[6-bp Tn5 barcode]AGATCGGAAGAGCACACGTC -3'
                                    3'- GATGTGCTGCGAGAAGGCTAGA[16-bp GEM barcode][10-bp UMI]AAAGAATATACCCXXXXXXXXX...XXXXXXXXXGACAGAGAATATGTGTAGA[6-bp Tn5 barcode]TCTAGCCTTCTCGTGTGCAG -5'

(6) cDNA purification:


5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp GEM barcode][10-bp UMI]TTTCTTATATGGGXXXXXXXXX...XXXXXXXXXCTGTCTCTTATACACATCT[6-bp Tn5 barcode]AGATCGGAAGAGCACACGTC -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp GEM barcode][10-bp UMI]AAAGAATATACCCXXXXXXXXX...XXXXXXXXXGACAGAGAATATGTGTAGA[6-bp Tn5 barcode]TCTAGCCTTCTCGTGTGCAG -5'

(7) Add S5R-P5 and S-P7-index primer for library amplification:


5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-------->
5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp GEM barcode][10-bp UMI]TTTCTTATATGGGXXXXXXXXX...XXXXXXXXXCTGTCTCTTATACACATCT[6-bp Tn5 barcode]AGATCGGAAGAGCACACGTC -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp GEM barcode][10-bp UMI]AAAGAATATACCCXXXXXXXXX...XXXXXXXXXGACAGAGAATATGTGTAGA[6-bp Tn5 barcode]TCTAGCCTTCTCGTGTGCAG -5'
                                                                                                                                             <---------------------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG[6-bp i7]TAGAGCATACGGCAGAAGACGAAC -5'

(8) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGXXX...XXXCTGTCTCTTATACACATCTNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCXXX...XXXGACAGAGAATATGTGTAGANNNNNNTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   Truseq Read 1               16-bp       10 bp                   cDNA          ME          6-bp          Truseq Read 2             6-bp        Illumina P7
                                                                 GEM barcode     UMI                                          Tn5 barcode                             Sample Index

Library sequencing:

(1) Add TruSeq Read 1 primer to sequence the first read (bottom strand as template, sequence 16-bp GEM barcode and 10-bp UMI, spacer and cDNA, 150 cycles):


                         5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT----------------------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCXXX...XXXGACAGAGAATATGTGTAGANNNNNNTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

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


                                                                                                                                    5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC----->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCXXX...XXXGACAGAGAATATGTGTAGANNNNNNTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add TruSeq Read 2 primer to sequence the second read (top strand as template, sequence Tn5 barcode and cDNA, 150 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGXXX...XXXCTGTCTCTTATACACATCTNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                        <------------------------------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'