The detailed step-by-step experimental protocols of Quartz-seq and Quartz-seq2 can be found at the RIKEN's website. Quartz-seq cosntructs the library of each single cell spearately, so it has low plexity. The indices are from Illumina Truseq adapters. The presence of T7 promoter sequence is only for Quartz-chip. Quartz-seq2 increased the throughput by using barcoded RT primer (1536 barcoded RT primers) to profile 3' end of cDNA. The cell barcode sequence can be found here.
RT primer (WTA): 5'-
Tagging primer: 5'-
Suppression primer: 5′- (NH2)-G
TRSU: 5'-
TRSI (indexed oligo): 5'-
Prepare indexed Truseq adapters by annealing TRSU and TRSI (formed the Y-shaped adapters):
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGAC GCTCTTCCGATC*T -3'CGAGAAGGCTAG 3'-G*TTCGTCTTCTGCCGTATGCTCTA NNNNNNCACTGACCTCAAGTCTGCACA
TPC1: 5'-
TPC2: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
TruSeq Read 1 sequencing primer: 5'-
Index 1 sequencing primer: 5'-
TruSeq Read 2 sequencing primer: 5'-
5'- XXXXXXXXXXXXXXXXXX(A)n <----(T)24GC GGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5'
1. RT product (DNA-RNA hybrid): 5'- XXXXXXXXXXXXXXXXXXXXX(A)n XXXXXXXXXXXXXXXXXXXXX(T)24GC GGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5' 2. RT primer leftover: 3'- (T)24GCGGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5'
1. From RT product: 3'- (pA)XXXXXXXXXXXXXXXXXXXXX(T)24GC GGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5' 2. From RT primer leftover: 3'- (pA)(T)24GCGGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5'
1. From RT product: 5'- TATAGAATTCGCGGCCGCTCGCGA (T)24----------> <----------(pA)XXXXXXXXXXXXXXXXXXXXX(dT)24GCGGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5' 2. From RT primer leftover: 5'-TATAGAATTCGCGGCCGCTCGCGA (T)24----------> <----------(pA)(T)24GCGGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5'
1. From RT product (amplifiable, see the next step): 5'- TATAGAATTCGCGGCCGCTCGCGA (dT)XXXXX...XXXXX(pA)CGCCCTATAGTGAGTCGTATTA TCGCGAGCGGCCGCGAATTCTATA ATATCTTAAGCGCCGGCGAGCGCT (pA)XXXXX...XXXXX(dT)GCGGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5' 2. From RT primer leftover (not amplifiable due to semi-suppressive PCR, omitted in the next step): 5'-TATAGAATTCGCGGCCGCTCGCGA (dT)(pA)CGCCCTATAGTGAGTCGTATTA TCGCGAGCGGCCGCGAATTCTATA ATATCTTAAGCGCCGGCGAGCGCT (pA)(dT)GCGGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5'
5′- (NH2)-G TATAGAATTCGCGGCCGCTCGCGA T----------> 5'-TATAGAATTCGCGGCCGCTCGCGA (dT)XXXXX...XXXXX(pA)CGCCCTATAGTGAGTCGTATTA TCGCGAGCGGCCGCGAATTCTATA ATATCTTAAGCGCCGGCGAGCGCT (pA)XXXXX...XXXXX(dT)GCGGGATATCACTCAGCATAAT AGCGCTCGCCGGCGCTTAAGATAT -5' <---------TAGCGCTCGCCGGCGCTTAAGATAT G-(NH2) -5'
Product 1 (left end plus cDNA, cannot be fully ligated due to 5' blocked by NH2, omitted in the next step): 5'- (NH2)-G TATAGAATTCGCGGCCGCTCGCGA (dT)XXXXX...XXXXXA -3' 3'- ACATATCTTAAGCGCCGGCGAGCGCT (pA)XXXXX...XXXXX -5' Product 2 (right end plus cDNA, cannot be fully ligated due to 5' blocked by NH2, omitted in the next step): 5'- (NH2)-GTATAGAATTCGCGGCCGCTCGCGA TAATACGACTCACTATAGGG CG(dT)XXXXX...XXXXXA -3' 3'- ACATATCTTAAGCGCCGGCGAGCGCT ATTATGCTGAGTGATATCCC GC(pA)XXXXX...XXXXX -5' Product 3 (middle of cDNA, can be fully ligated, hence only sequence-able fragments): 5'- XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXA -3' 3'- AXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXX -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGAC ACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'GCTCTTCCGATCT XXXXXXXXXXX...XXXXXXXXXXXAGATCGGAAGAGC CGAGAAGGCTAG AXXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCG 3'-GTTCGTCTTCTGCCGTATGCTCTA NNNNNNCACTGACCTCAAGTCTGCACA CAGCACATCCCTTTCT CACATCTAGAGCCACCAGCGGCATAGTAA -5'
Top strand will be amplified like this: 5'- AATGATACGGCGACCACCGAG ----------> 5'-AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXXXXXXXXXX...XXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <----------GAGCATACGGCAGAAGACGAAC -5'Bottom strand will be amplified like this: 5'- CAAGCAGAAGACGGCATACGAGAT ----------> 3'-GTTCGTCTTCTGCCGTATGCTCTA NNNNNNCACTGACCTCAAGTCTGCACACGAGAAGGCTAG AXXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCGCAGCACATCCCTTTCT CACATCTAGAGCCACCAGCGGCATAGTAA -5' <----------GAGCCACCAGCGGCATAGTAA -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXXXXXXXXXX...XXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAG AXXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 TruSeq Read 1 cDNATruSeq Read 2 cellIllumina P7 index
5'- ACAC TCTTTCCCTACACGACGCTCTTCCGATCT ----------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAG AXXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAG AXXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXXXXXXXXXX...XXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <----------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG
eMDRT0001 - eMDRT1536: 5'-
Tagging primer: 5'-
gM primer (the same as Suppression primer in Quartz-seq): 5′- G
rYshapeP5: 5'-
rYshapeP7LT (Sample Index Oligos): 5'-
Prepare truncated sequence adapter indexed Truseq adapters by annealing rYshapeP5 and rYshapeP7LT:
5'- CAAGCAGAAGACGGCATACGAGAT [6-bp sample index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3'CGAGAAGGCTAG -5' 3'-ATGTGCTG
P5-gMac_hybrid: 5'-
TPC2: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
Read1DropQuartz primer: 5'-
Index 1 sequencing primer: 5'-
TruSeq Read 2 sequencing primer: 5'-
5'- TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (T)24----------> (A)nXXXXXXXXXXXXXXXXXXXXX -5'
1. RT product (DNA-RNA hybrid): 5'- TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXXXXXXXXXXXXXXXXXX (pA)XXXXXXXXXXXXXXXXXXXXX -5' 2. RT primer leftover: 5'-TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT) -3'
1. From RT product: 5'- TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXXXXXXXXXXXXXXXXXX(pA) -3' 2. From RT primer leftover: 5'-TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)(pA) -3'
1. From RT product: 5'- TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXX...XXXXX(pA)----------> <----------(dT)AGCGCTCGCCGGCGCTTAAGATAT -5' 2. From RT primer leftover: 5'-TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)(pA)----------> <----------(dT)AGCGCTCGCCGGCGCTTAAGATAT -5'
1. From RT product (amplifiable, see the next step): 5'- TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXX...XXXXX(pA)TCGCGAGCGGCCGCGAATTCTATA ATATCTTAAGCGCCGGCGAGCGCTA TG[15-bp cell barcode] [8-bp UMI] (pA)XXXXX...XXXXX(dT)AGCGCTCGCCGGCGCTTAAGATAT -5' 2. From RT primer leftover (not amplifiable due to semi-suppressive PCR, omitted in the next step): 5'-TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)(pA)TCGCGAGCGGCCGCGAATTCTATA ATATCTTAAGCGCCGGCGAGCGCTA TG[15-bp cell barcode] [8-bp UMI] (pA)(dT)AGCGCTCGCCGGCGCTTAAGATAT -5'
5′- G TATAGAATTCGCGGCCGCTCGCGA T----------> 5'-TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXX...XXXXX(pA)TCGCGAGCGGCCGCGAATTCTATA ATATCTTAAGCGCCGGCGAGCGCTA TG[15-bp cell barcode] [8-bp UMI] (pA)XXXXX...XXXXX(dT)AGCGCTCGCCGGCGCTTAAGATAT -5' <---------TAGCGCTCGCCGGCGCTTAAGATAT G -5'
Product 1 (eMDRT plus 3'-end of cDNA): 5'- G TATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXX...XXXXXA ACATATCTTAAGCGCCGGCGAGCGCTA TG[15-bp cell barcode] [8-bp UMI] (pA)XXXXX...XXXXX -5' Product 2 (Tagging primer plus 5'-end cDNA): 5'- GTATAGAATTCGCGGCCGCTCGCGA (dT)XXXXX...XXXXXA -3' ACATATCTTAAGCGCCGGCGAGCGCT (pA)XXXXX...XXXXX -5' Product 3 (middle of cDNA): 5'- XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXA -3' 3'- AXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXX -5'
Product 1 (eMDRT plus 3'-end of cDNA, the only amplifiable fragments, see the next step) GTCGTGTA 5'-CAAGCAGAAGACGGCATACGAGAT [6-bp sample index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT GTATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXX...XXXXXAGATCGGAAGAGC CGAGAAGGCTAG ACATATCTTAAGCGCCGGCGAGCGCTA TG[15-bp cell barcode] [8-bp UMI] (pA)XXXXX...XXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [6-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'ATGTGCTG Product 2 (Tagging primer plus 5'-end cDNA, cannot be amplified due to primers used, see the next step clarification) GTCGTGTA 5'-CAAGCAGAAGACGGCATACGAGAT [6-bp sample index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT GTATAGAATTCGCGGCCGCTCGCGA (dT)XXXXX...XXXXXAGATCGGAAGAGC CGAGAAGGCTAG ACATATCTTAAGCGCCGGCGAGCGCT (pA)XXXXX...XXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [6-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'ATGTGCTG Product 3 (middle of cDNA, cannot be amplified due to primers used, omitted in the next step) GTCGTGTA 5'-CAAGCAGAAGACGGCATACGAGAT [6-bp sample index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXAGATCGGAAGAGC CGAGAAGGCTAG AXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [6-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'ATGTGCTG
Product 1 (eMDRT plus 3'-end of cDNA) will be amplified like this: 5'- AATGATACGGCGACCACCGAGATCTACA T TGTATAGAATTCGCGGCCGCTCGCGAT AC---------->GTCGTGTA 5'-CAAGCAGAAGACGGCATACGAGAT [6-bp sample index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT GTATAGAATTCGCGGCCGCTCGCGAT AC[15-bp cell barcode] [8-bp UMI] (dT)XXXXX...XXXXXAGATCGGAAGAGC CGAGAAGGCTAG ACATATCTTAAGCGCCGGCGAGCGCTA TG[15-bp cell barcode] [8-bp UMI] (pA)XXXXX...XXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [6-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'ATGTGCTG <----------GAGCATACGGCAGAAGACGAAC -5'Product 2 (Tagging primer plus 5'-end cDNA) cannot be amplified, note the P5-gMac primer ends with "AC" which will not anneal to the template: 5'- AATGATACGGCGACCACCGAGATCTACA T TGTATAGAATTCGCGGCCGCTCGCGAT ACGTCGTGTA 5'-CAAGCAGAAGACGGCATACGAGAT [6-bp sample index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT GTATAGAATTCGCGGCCGCTCGCGA (dT)XXXXX...XXXXXAGATCGGAAGAGC CGAGAAGGCTAG ACATATCTTAAGCGCCGGCGAGCGCT (pA)XXXXX...XXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [6-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'ATGTGCTG GAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACA TTGTATAGAATTCGCGGCCGCTCGCGAT ACNNNNNNNNNNNNNNN NNNNNNNN (dT)XXXXXXXXXXX...XXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGT AACATATCTTAAGCGCCGGCGAGCGCTA TGNNNNNNNNNNNNNNN NNNNNNNN (pA)XXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 15-bp 8bp UMI cDNATruSeq Read 2 6bpIllumina P7 cell barcode sample index
5'- ACA TTGTATAGAATTCGCGGCCGCTCGCGAT AC----------------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGT AACATATCTTAAGCGCCGGCGAGCGCTA TGNNNNNNNNNNNNNNN NNNNNNNN (pA)XXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGT AACATATCTTAAGCGCCGGCGAGCGCTA TGNNNNNNNNNNNNNNN NNNNNNNN (pA)XXXXXXXXXXX...XXXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACA TTGTATAGAATTCGCGGCCGCTCGCGAT ACNNNNNNNNNNNNNNN NNNNNNNN (dT)XXXXXXXXXXX...XXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <----------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG