10x Chromium 5' Immune Profiling Feature Barcoding

As the technology keeps developing, there are quite a few different kits now on the 10x Genomics website. I recommend you contact them to choose the appropriate kit for your application. In this page, the v2 version of The Chromium Single Cell 5' Immune Profiling Feature Barcoding is shown here. The workflow is based on their RevA version of the user guide. This kit can profile gene expression (5'), VDJ of BCR and TCR and surface protein. If you only need one of them, the basic principle is the same. All three layers of information is shown here to give an overview. The 5' kit is similar to the 3' kit. Instead of using barcoded RT primers on the beads, the 5' kit uses the same RT primer, but with barcoded Template Switching Oligos (TSO) on their gel beads. The antibody against surface proteins has a barcoded oligo that is reverse complement to the TSO on the beads, which will also be captured inside the droplet. Conceptually, this kit is very similar to STRT-seq. The 16-bp cell barcodes are the same as the 3' Gene Expression kit v2, and you can download from here: 737K-august-2016.txt.gz. This file is copied from Cell Ranger (using Cell Ranger v2.1.0 as an example) /path/to/cellranger-2.1.0/cellranger-cs/2.1.0/tenkit/lib/python/tenkit/barcodes.

For the VDJ part, the kit basically uses a combination of loci specific (VDJ of B or T cells) primer mix to perform a nested PCR to get the information of VDJ. The sequence of those loci specific primer mix can be found in the user guide. If you blat/map them, you will see they anneal to the constant region of the genes. I'm not an expert on this, so I'm not able to comment on the details of these primers.

Around 2023-2024, the v3 chemistry was introduced. It has better cell recovery and sensitivity (number of detected genes per cell and TRA/TRB UMI counts) compared to v1 and v2. See their product sheet for a more detailed overview. The cell barcodes have changed in the v3 chemistry, and the file with all 16-bp cell barcodes can be found here: 3M-5pgex-jan-2023.txt.gz. This file is copied from Cell Ranger (using Cell Ranger v8.0.1 as an example) /path/to/cellranger-8.0.1/lib/python/cellranger/barcodes/. The library structure of 5' v3 is exactly the same as 5' v1 and v2, except that the UMI is 10-bp long in v1 and v2 but 12-bp in v3.

Adapter and primer sequences:

Next GEM Single Cell 5' Gel Beads:


          V1 & V2 (PN-1000264 or PN-1000267): |--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATrGrGrG -3'
   
                             V3 (PN-2001129): |--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][12-bp UMI]TTTCTTATATrGrGrG -3'

Poly-dT RT primer (PN-2000007): 5'- AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN -3'

Barcoded oligo (FeatureBarcode, FB) on antibody against surface protein: 5'- CGGAGATGTGTATAAGAGACAGNNNNNNNNNN[15-bp FB]NNNNNNNNNCCCATATAAGAAA -3'

Feature cDNA Primers 4 (for cDNA amplification, PN-2000277, these are a mixture of three primers):


        Forward primer: 5'- CTACACGACGCTCTTCCGATCT -3'

        Reverse primer (for cDNA): 5'- AAGCAGTGGTATCAACGCAGAG -3'

        Reverse primer (for FB):  5'- CTCGTGGGCTCGGAGATGTG -3'

The foward primer of T/B Mix 1 & 2 is the same: 5'- GATCTACACTCTTTCCCTACACGACGC -3'

For reverse primers of T/B cell Mix 1 (PN-2000242, 2000254, 2000256, 2000258): check the user guide for the sequence details. These are the outer primers.

For reverse primers of T/B cell Mix 2 (PN-2000246, 2000255, 2000257, 2000259): check the user guide for the sequence details. These are the inner primers.

Truseq adapter (double stranded DNA with a T overhang, PN-220026):


        5'-  GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
        3'- TCTAGCCTTCTCG -5'

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

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

Illumina Nextera Read 2 primer: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'

Dual Index Kit TT Set A (PN-3000431):


         Forward primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp i5]ACACTCTTTCCCTACACGACGCTC -3'

         Reverse primer: 5'- CAAGCAGAAGACGGCATACGAGAT[10-bp i7]GTGACTGGAGTTCAGACGTGT -3'

Dual Index Kit TN Set A (PN-3000510):


         Forward primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp i5]ACACTCTTTCCCTACACGACGCTC -3'

         Reverse primer: 5'- CAAGCAGAAGACGGCATACGAGAT[10-bp i7]GTCTCGTGGGCTCGG -3'

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

Nextera Sample index sequencing primer: 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'

Truseq i5 index sequencing primer (index2): 5'- AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Step-by-step library generation

(1) Reverse transcription with Poly-dT RT primer using MMLV:


1.1) mRNA:

                   <--------NV(T)₃₀CATGAGACGCAACTATGGTGACGAA -5'
    5'- XXXXXXXXXXXXXXXXXXXXXB(A)₃₀

1.2) Feature (un-affected):

    3'- AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGC -5'

(2) The terminal tranferase acitivity of MMLV adds extra Cs:


2.1) mRNA:

     CCCXXXXXXXXXXXXXXXXXXXXXNV(T)₃₀CATGAGACGCAACTATGGTGACGAA -5'
    5'- XXXXXXXXXXXXXXXXXXXXXXB(A)₃₀

2.2) Feature (un-affected):

    3'- AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGC -5'

(3) cDNA and Feature capture by gel bead barcoded TSO:


3.1) cDNA:
                                                          <-----------CCCXXXXXXXXXXXXXXXXXXXXXNV(T)₃₀CATGAGACGCAACTATGGTGACGAA -5'
|--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXXXXXXXXXXXXXXXXXXXXXB(A)₃₀------->

3.2) Feature:

                                                <-----------AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGC -5'
|--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGG------->

(4) Thhere are two products after GEM-RT: the cDNA is long and the Feature is short:


4.1) cDNA (long):

|--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXXXXXXXXXXXXXXXXXXXXXB(pA)GTACTCTGCGTTGATACCACTGCTT -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXXXXXXXXXXXXXXXXXXXXNV(dT)CATGAGACGCAACTATGGTGACGAA -5'

4.2) Feature (short):

|--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGNNNNNNNNN[15-bp FB]NNNNNNNNNNCTGTCTCTTATACACATCTCCG -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGC -5'

(5) Purify GEM-RT products and add Feature cDNA Primers 4 (PN-2000277) to amplify cDNA and Feature together:


5.1) cDNA (long):

   5'- CTACACGACGCTCTTCCGATCT-------------->
|--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXXXXXXXXXXXXXXXXXXXXXB(pA)GTACTCTGCGTTGATACCACTGCTT -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXXXXXXXXXXXXXXXXXXXXNV(dT)CATGAGACGCAACTATGGTGACGAA -5'
                                                                                     <-----------------GAGACGCAACTATGGTGACGAA -5'

5.2) Feature (short):

   5'- CTACACGACGCTCTTCCGATCT-------------->
|--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGNNNNNNNNN[15-bp FB]NNNNNNNNNNCTGTCTCTTATACACATCTCCG -3'
   3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGC -5'
                                                                                                  <---------------GTGTAGAGGCTCGGGTGCTC-5'

(6) Use SPRI Select size selection to physically separate long (cDNA) and short (Feature) fragments for library preparation separately:


6.1) cDNA (long):

5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXXXXXXXXXXXXXXXXXXXXXB(pA)GTACTCTGCGTTGATACCACTGCTT -3'
3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXXXXXXXXXXXXXXXXXXXXNV(dT)CATGAGACGCAACTATGGTGACGAA -5'

6.2) Feature (short):

5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGNNNNNNNNN[15-bp FB]NNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAG -3'
3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTC -5'

(7) Use Dual Index Kit TN Set A (PN-3000510) primers to make Feature library:


5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp i5]ACACTCTTTCCCTACACGACGCTC----------------->
                                                  5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGNNNNNNNNN[15-bp FB]NNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAG -3'
                                                  3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTC -5'
                                                                                                                                                       <----------------GGCTCGGGTGCTCTG[10-bp i7]TAGAGCATACGGCAGAAGACGAAC -5'

(8) Purify Feature library which is ready to sequence:


5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGNNNNNNNNN[15-bp FB]NNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAGAC[10-bp i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[10-bp i5]TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCNNNNNNNNN[15-bp FB]NNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[10-bp i7]TAGAGCATACGGCAGAAGACGAAC -5'

(9) For cDNA part, if gene expression is of interest, check this page for details, and the gene expression library will be ommited here. For immune profiling of VDJ, add T/B Cell Mix 1 (Forward and outer primers) for the first round of amplification:


5'- GATCTACACTCTTTCCCTACACGACGC-------------->
                5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXX...-V-D-J-[the constant regions]...XXX(pA)GTACTCTGCGTTGATACCACTGCTT -3'
                3'- GATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXX...-V-D-J-[the constant regions]...XXX(dT)CATGAGACGCAACTATGGTGACGAA -5'
                                                                                                  <-------[outer primers] -5'

(10) Purify the product and add T/B Cell Mix 2 (Forward and inner primers) for the second round of amplification:


5'- GATCTACACTCTTTCCCTACACGACGC-------------->
5'- GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXX...-V-D-J-[the constant regions] -3'
3'- CTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXX...-V-D-J-[the constant regions] -5'
                                                                                           <-------[inner primers] -5'

(11) Purify the product after the second round of PCR:


5'- GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXX...-V-D-J-[inner primers] -3'
3'- CTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXX...-V-D-J-[inner primers] -5'

(12) Fragment the product and A tailing:


Product 1 (left part):

5'- GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXX...-V-D-J-...XXX*A -3'
3'- CTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXX...-V-D-J-...XXX   -5'

Product 2 (the rest):

5'-   XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXX*A -3'
3'- A*XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXX   -5'

(13) Truseq adapter (PN-220026) ligation:


Product 1 (left part):

5'- GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXX...-V-D-J-...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
3'- CTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXX...-V-D-J-...XXXTCTAGCCTTCTCG -5'

Product 2 (the rest, not amplifiable, will be ommitted):

5'-                      GCTCTTCCGATCTXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
3'- CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXTCTAGCCTTCTCG -5'

(14) Use Dual Index Kit TT Set A (PN-3000431) primers to make VDJ library:


5'- AATGATACGGCGACCACCGAGATCTACAC[10-bp i5]
                                           ACACTCTTTCCCTACACGACGCTC---------->
                                  5'- GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[16-bp cell barcode][10-bp UMI]TTTCTTATATGGGXXX...-V-D-J-...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
                                  3'- CTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[16-bp cell barcode][10-bp UMI]AAAGAATATACCCXXX...-V-D-J-...XXXTCTAGCCTTCTCG -5'
                                                                                                                                         <--------------TGTGCAGACTTGAGGTCAGTG[10-bp i7]TAGAGCATACGGCAGAAGACGAAC -5'

(15) Final library structure:

(15.1) Gene expression library (click here to see how it is generated):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNN..NNNNNTTTCTTATATGGGXXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNN..NNNNNAAAGAATATACCCXXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5              10 bp              Truseq Read 1               16 bp     10 or 12 bp                 cDNA          Truseq Read 2                10 bp        Illumina P7
                                  i5 index                                    cell barcode      UMI                                                           Sample Index

(15.2) Feature library (the 15-bp FeatureBarcode tells you the identify of the protein):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNN..NNNNNTTTCTTATATGGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNN..NNNNNAAAGAATATACCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
              Illumina P5          10 bp           Truseq Read 1                  16 bp     10 or 12 bp                 9 bp       15 bp        10 bp            Nextera Read 2             10 bp         Illumina P7
                                 i5 index                                     cell barcode      UMI                    spacer  FeatureBarcode  spacer                                    Sample Index

(15.3) Immune Profiling VDJ library:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNN..NNNNNTTTCTTATATGGGXXX...-V-D-J-...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNN..NNNNNAAAGAATATACCCXXX...-V-D-J-...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5              10 bp              Truseq Read 1               16 bp     10 or 12bp                       VDJ                Truseq Read 2               10 bp        Illumina P7
                                  i5 index                                    cell barcode      UMI                                                                      Sample Index

Library sequencing:

(1) Add Truseq Read 1 primer to sequence the first read (bottom strand as template, sequence 16-bp cell barcode and UMI, 26 cycles for V1 and V2, 28 cycles for V3):


(i) Gene expression library:
                                       5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCXXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(ii) Feature library:
                                       5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(iii) Immune Profiling VDJ library:
                                       5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCXXX...-V-D-J-...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Sample Index sequencing primer to sequence the sample index (bottom strand as template, 10 cycles):


(i) Gene expression library:
                                                                                                                         5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCXXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(ii) Feature library:
                                                                                                                                                 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(iii) Immune Profiling VDJ library:
                                                                                                                                   5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC--------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNAAAGAATATACCCXXX...-V-D-J-...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Truseq i5 index sequencing primer (index2) to sequence i5 index (top strand as template, 10 cycles):


(i) Gene expression library:
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGXXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <---------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -5'

(ii) Feature library:
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <---------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -5'

(iii) Immune Profiling VDJ library:
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGXXX...-V-D-J-...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <---------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -5'

(4) Add Read 2 sequencing primers to sequence read 2 (top strand as template, 90 cycles):


(i) Gene expression library:
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGXXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                                  <---------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'

(ii) Feature library:
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                                                           <---------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(iii) Immune Profiling VDJ library:
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTATATGGGXXX...-V-D-J-...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                                            <---------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'