# SMART-seq / SMART-seq2

The protocols of SMART-seq and SMART-seq2 are almost the same. SMART-seq2 is an improved version of SMART-seq. The authors performed 457 optimisation experiments to test conditions. Two key parameters are:

(1) exchanging the last guanylate for a locked nucleic acid (LNA) at the 3' end of TSO.

(2) Include methyl group donor betaine in combination with higher MgCl2 concentrations.

## Adapter and primer sequences:

oligo-dTVN: 5'- AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN -3'

Template Switching Oligo (TSO): 5'- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G -3'

ISPCR: 5′- AAGCAGTGGTATCAACGCAGAGT -3′

Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'- AGATGTGTATAAGAGACAG -3'

Nextera N/S5xx primer entry point (s5): 5'- TCGTCGGCAGCGTC -3'

Nextera N7xx primer entry point (s7): 5'- GTCTCGTGGGCTCGG -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Nextera (XT) N/S5xx Index primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[8-bp i5 index]TCGTCGGCAGCGTC -3'

Nextera (XT) N7xx Index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp i7 index]GTCTCGTGGGCTCGG -3'

Read 1 sequencing primer: 5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG -3'

Index 1 sequencing primer: 5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3'

Read 2 sequencing primer: 5'- GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG -3'

### 8-bp i5 & i7 sequences:

        N/S502 : CTCTCTAT
        N/S503 : TATCCTCT
        N/S505 : GTAAGGAG
        N/S506 : ACTGCATA
        N/S507 : AAGGAGTA
        N/S508 : CTAAGCCT
        N/S510 : CGTCTAAT
        N/S511 : TCTCTCCG
        N/S513 : TCGACTAG
        N/S515 : TTCTAGCT
        N/S516 : CCTAGAGT
        N/S517 : GCGTAAGA
        N/S518 : CTATTAAG
        N/S520 : AAGGCTAT
        N/S521 : GAGCCTTA
        N/S522 : TTATGCGA

        N701 : TCGCCTTA
        N702 : CTAGTACG
        N703 : TTCTGCCT
        N704 : GCTCAGGA
        N705 : AGGAGTCC
        N706 : CATGCCTA
        N707 : GTAGAGAG
        N710 : CAGCCTCG
        N711 : TGCCTCTT
        N712 : TCCTCTAC
        N714 : TCATGAGC
        N715 : CCTGAGAT
        N716 : TAGCGAGT
        N718 : GTAGCTCC
        N719 : TACTACGC
        N720 : AGGCTCCG
        N721 : GCAGCGTA
        N722 : CTGCGCAT
        N723 : GAGCGCTA
        N724 : CGCTCAGT
        N726 : GTCTTAGG
        N727 : ACTGATCG
        N728 : TAGCTGCA
        N729 : GACGTCGA

## Step-by-step library generation

### (1) Anneal oligo-dTVN to mRNA and reverse transcription using MMLV:

    5'- XXXXXXXXXXXXXXXXXXXB(A)n
                     <----NV(T)30CATGAGACGCAACTATGGTGACGAA -5'

### (2) After reverse transcription, the terminal tranferase acitivity of MMLV add extra Cs:

    5'- XXXXXXXXXXXXXXXXXXXXB(A)n
     CCCXXXXXXXXXXXXXXXXXXXNV(T)30CATGAGACGCAACTATGGTGACGAA -5'

### (3) Adding TSO for second strand synthesis:

    5'- AAGCAGTGGTATCAACGCAGAGTACATGGGXXXXXXXXXXXXXXXXXXXX(A)n
                            <------CCCXXXXXXXXXXXXXXXXXXXX(T)30CATGAGACGCAACTATGGTGACGAA -5'

### (4) Adding ISPCR for single primer cDNA amplification:( i.e. semi-suppressive PCR )

    5'- AAGCAGTGGTATCAACGCAGAGT------>
    5'- AAGCAGTGGTATCAACGCAGAGTACATGGGXXXXXXX...XXXXXXX(pA)GTACTCTGCGTTGATACCACTGCTT
        TTCGTCACCATAGTTGCGTCTCATGTACCCXXXXXXX...XXXXXXX(dT)CATGAGACGCAACTATGGTGACGAA -5'
                                                      <------TGAGACGCAACTATGGTGACGAA -5'

### (5) Nextera tagmentation on amplified cDNA (will create 9-bp gap):

    Product 1 (s5 at both ends, not amplifiable due to semi-suppressiev PCR):

    5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                      TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'

    Product 2 (s7 at both ends, not amplifiable due to semi-suppressiev PCR):

    5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                       TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

    Product 3 (different ends, amplifiable):

    5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                      TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

### (6) 72 degree gap fill-in (the first cycle in Nextera PCR):

    5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
        AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

### (7) Amplification using N/S5xx and N7xx index primers:

    5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC---->
                                     5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC
                                         AGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                   <----GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

### (8) Final library structure:

    5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
        TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
               Illumina P5              i5         s5              ME                cDNA                ME               s7          i7            Illumina P7

## Library sequencing:

### (1) Add read 1 sequencing primer to sequence the first read (bottom strand as template):

                                         5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG------>
    3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

### (2) Add index 1 sequencing primer to sequence the first index (i7) (bottom strand as template):

                                                                                             5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------>
    3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

### (3) Folds over and sequence the second index (i5) (bottom strand as template):

    5'- AATGATACGGCGACCACCGAGATCTACAC------>
        TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

### (4) Cluster regeneration, add read 2 sequencing primer to sequence the second read (top strand as template):

    5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                                                                          <------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'