PIP-seq V1 Prototype

This library structure was only used in some of the samples, such as SRR19086115, SRR19086119 and a few additional samples in the gefitinibis response experiments. The beads generation procedures should be similar to inDrop and Delley2021. There is very little information about this library structure, so this page is based on educational guesses.

Adapter and primer sequences:

Sequence used during the experiment:

* Barcoded beads-oligo: |--5'- /5Acryd/TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[0 - 3bp Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI]TTTTTTTTTTTTTTTTTTTV -3'

PIPS_TSO: 5'- AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG -3'

PIPS_WTA_primer: 5'- AAGCAGTGGTATCAACGCAGAGT -3'

PIPs_P5library: 5'- AATGATACGGCGACCACCGAGATCTACACTAGATCGCCTCTTTCCCTACACGACGC -3'

Nextera N7xx: 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp i7 index]GTCTCGTGGGCTCGG -3'

TruSeq Read 1: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'

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

Nextera Read 2: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

* The 0 - 3 bp Spacer is None or A or GA or TGA.

Step-by-step library generation

(1) Cell encapsulation by vortexing, cell lysis by heat, mRNA capture, then add RT reagent for reverse transcription:


|--5'- /5Acryd/TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI]TTTTTTTTTTTTTTTTTTTV---->
                                                                                                                                                           AAAAAAA...AAAAAABXXX...XXX -5'

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


|--5'- /5Acryd/TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI](dT)VXXX...XXXCCC
                                                                                                                                                        (pA)BXXX...XXX -5'

(3) TSO is already in the RT reagent and it will incorporate into the template:


|--5'- /5Acryd/TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI](dT)VXXX...XXXCCC----->
                                                                                                                                                        (pA)BXXX...XXXGGGTAAGTGAGACGCAACTATGGTGACGAA -5'

(4) This is the first-strand cDNA after reverse transcription:


|--5'- /5Acryd/TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI](dT)VXXX...XXXCCCATTCACTCTGCGTTGATACCACTGCTT -3'

(5) Without purification, immediately add PIPS_WTA_primer for single-primer semi-suppressive PCR:


                  5'- AAGCAGTGGTATCAACGCAGAGT------------------>
|--5'- /5Acryd/TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI](dT)VXXX...XXXCCCATTCACTCTGCGTTGATACCACTGCTT -3'
                                                                                                                                                        <--------------------TGAGACGCAACTATGGTGACGAA -5'

(6) Purify amplified double-stranded cDNA::


5'- AAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI](dT)VXXX...XXXCCCATTCACTCTGCGTTGATACCACTGCTT -3'
3'- TTCGTCACCATAGTTGCGTCTCATGCTGAGGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[Spacer][8-bp barcode1]CTCACTAACGAACACTGCGGAA[8-bp barcode2][6-bp UMI](pA)BXXX...XXXGGGTAAGTGAGACGCAACTATGGTGACGAA -5'

(7) Use the Illumina Nextera XT kit for cDNA fragmentation:


 Product 1 (left end of cDNA + Nextera s7, the only amplifiable fragment):

5'- AAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI](dT)VXXX...XXX         CTGTCTCTTATACACATCT -3'
3'- TTCGTCACCATAGTTGCGTCTCATGCTGAGGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[Spacer][8-bp barcode1]CTCACTAACGAACACTGCGGAA[8-bp barcode2][6-bp UMI](pA)BXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'


 Product 2 (right end of cDNA + Nextera s7, not amplifiable):

5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXCCCATTCACTCTGCGTTGATACCACTGCTT
                   TCTACACATATTCTCTGTC         XXX...XXXGGGTAAGTGAGACGCAACTATGGTGACGAA -3'


 Products 3 - 7 (omitted, none of them are amplifiable due the primers used in the next round):

    Left end of cDNA + Nextera s5
    Right end of cDNA + Nextera s5
    Nextera s5 or s7 + middle part of the cDNA + Nextera s5 or s7

(8) Library Amplification using PIPs_P5library and Nextera N7xx primers:


5'- AATGATACGGCGACCACCGAGATCTACACTAGATCG
                                        CCTCTTTCCCTACACGACGC------------------>
       5'- AAGCAGTGGTATCAACGCAGAGTACGACTCCTCTTTCCCTACACGACGCTCTTCCGATCT[Spacer][8-bp barcode1]GAGTGATTGCTTGTGACGCCTT[8-bp barcode2][6-bp UMI](dT)VXXX...XXX         CTGTCTCTTATACACATCT -3'
       3'- TTCGTCACCATAGTTGCGTCTCATGCTGAGGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[Spacer][8-bp barcode1]CTCACTAACGAACACTGCGGAA[8-bp barcode2][6-bp UMI](pA)BXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                                                                        <--------------GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

(9) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACTAGATCGCCTCTTTCCCTACACGACGCTCTTCCGATCTN.NNNNNNNNNGAGTGATTGCTTGTGACGCCTTNNNNNNNNNNNNNN(dT)VXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGATCTAGCGGAGAAAGGGATGTGCTGCGAGAAGGCTAGAN.NNNNNNNNNCTCACTAACGAACACTGCGGAANNNNNNNNNNNNNN(pA)BXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
             Illumina P5                          TruSeq Read 1         ↓   8-bp          W1 linker       8-bp   6-bp        cDNA           ME               s7         8-bp        Illumina P7
                                                                    Spacer barcode1                     barcode2  UMI                                               sample index

Library sequencing using Illumina primers

(1) Add TruSeq Read 1 sequencing primer to sequence the first read (bottom strand as template, cell barcodes and UMI, at least 47 cycles):


                                  5'- ACAC
                                          TCTTTCCCTACACGACGCTCTTCCGATCT------------------------------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGATCTAGCGGAGAAAGGGATGTGCTGCGAGAAGGCTAGAN.NNNNNNNNNCTCACTAACGAACACTGCGGAANNNNNNNNNNNNNN(pA)BXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence the sample index at the i7 side (bottom strand as template, 8 cycles):


                                                                                                                                5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGATCTAGCGGAGAAAGGGATGTGCTGCGAGAAGGCTAGAN.NNNNNNNNNCTCACTAACGAACACTGCGGAANNNNNNNNNNNNNN(pA)BXXX...XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Nextera Read 2 primer to sequence the second read (top strand as template, >67 cycles, these are cDNA reads):


5'- AATGATACGGCGACCACCGAGATCTACACTAGATCGCCTCTTTCCCTACACGACGCTCTTCCGATCTN.NNNNNNNNNGAGTGATTGCTTGTGACGCCTTNNNNNNNNNNNNNN(dT)VXXX...XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                                             <------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'