CaRPool-seq library structure and sequencing

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Here, we'll describe the bcgRNA library construction step-by-step. Other resources have described in detail library construction for 10x Genomics 3' scRNA-seq chemistry, and CITE-seq and Cell Hashing.

RfxCas13d will process CRISPR array into individual mature crRNAs ending with a single detacble barcoded gRNA:

bcgRNA sequence:

5'- AACCCCUACCAACUGGUCGGGGUUUGAAACGCCUUGGCACCCGAGAAUUCCANNNNNNNNNNNNNNNGCUUUAAGGCCGGUCCUAGCAA[NNNNNNNNNN(N)_n] -3'

Adapter and primer sequences:

10x V3 3' kit bead oligos (there are three types of sequences):


       TruSeq Partial Read1 dT (for mRNA):
       |--5'- CTACACGACGCTCTTCCGATCT[16-bp cell barcode][12-bp UMI](T)₃₀ -3'

       Nextera Partial Read1 Capture Sequence 1 (for surface protein & CRISPR screening):
       |--5'- GTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGC -3'

       Nextera Partial Read1 Capture Sequence 2 (for CRISPR screening):
       |--5'- GTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]CCTTAGCCGCTAATAGGTGAGC -3'

  

Feature cDNA Primers 1 (PN-2000096, for CRISPR screening) contain two primers for sgRNA/bcgRNA amplification.
To improve bcgRNA amplification, we add an additive reverse primer (not provided in PN-2000096), similar to CITE-seq:
       Primers to amplify cDNA from bcgRNA:
       Forward primer: 5'- GCAGCGTCAGATGTGTATAAGAGACAG -3'
       Reverse primer: 5'- AAGCAGTGGTATCAACGCAGAG -3'
       Additive primer: 5'- CCTTGGCACCCGAGAATT^*C^*C -3'


bcgRNA library amplification primers [We'll use the forward primer from Feature SI Primers 1 (PN-2000099)]:
       Primers to index bcgRNA library:
       Forward primer: 5'- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
       Reverse primer: 5'- CAAGCAGAAGACGGCATACGAGAT[8bp sample index]GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA -3'
       


Template Switching Oligo (TSO): 5'- AAGCAGTGGTATCAACGCAGAGTACATrGrGrG -3'
Additive primer: 5'- CCTTGGCACCCGAGAATT^*C^*C -3'
Feature SI Primers 2 (PN-2000099): 5'- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
RPI i7 Sample Index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[i7]GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA -3'
Illumina Nextera Read 1 primer: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
Illumina smallRNA Read 2 primer: 5'- GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA -3'
Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'
Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'
Illumina P5 primer: 5'- AATGATACGGCGACCACCGA -3'
Illumina P7 primer: 5'- CAAGCAGAAGACGGCATACGAGA -3'

Step-by-step library generation

bcgRNA:

If using Capture Sequence 1:

(1) Reverse Transcription:


|--5'- GTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGC-------->
                                                            AACGAUCCUGGCCGGAAUUUCGNNNNNNNNNNNNNNNACCUUAAGAGCCCACGGUUCCCAAAGUUUGGGGCUGGUCAACCAUCCCCAA -5'
                                         3'- [_n(N)NNNNNNNNNN]

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

                                                 
|--5'- GTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGGTTTCAAACCCCGACCAGTTGGTAGGGGTTCCC -3'
                                                            AACGAUCCUGGCCGGAAUUUCGNNNNNNNNNNNNNNNACCUUAAGAGCCCACGGUUCCCAAAGUUUGGGGCUGGUCAACCAUCCCCAA    -5'
                                         3'- [_n(N)NNNNNNNNNN]

(3) TSO for template Switching:

Template switching may be inefficient due to terminal stem loop in template RNA.

                                                 
|--5'- GTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGGTTTCAAACCCCGACCAGTTGGTAGGGGTTCCC-------->
                                                            AACGAUCCUGGCCGGAAUUUCGNNNNNNNNNNNNNNNACCUUAAGAGCCCACGGUUCCCAAAGUUUGGGGCUGGUCAACCAUCCCCAAGGGTACATGAGACGCAACTATGGTGACGAA -5'
                                         3'- [_n(N)NNNNNNNNNN]

(4) Adding Feature cDNA Primers for cDNA amplification:

(i) using PN-2000096 F+R primers


5'- GCAGCGTCAGATGTGTATAAGAGACAG--------------->
  |--5'- GTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGGTTTCAAACCCCGACCAGTTGGTAGGGGTTCCCATGTACTCTGCGTTGATACCACTGCTT -3'
                                                                                                                                         <---------------TACATGAGACGCAACTATGGTGACGAA -5'

(ii) using PN-2000096 F and additive R primers (independent of template switching)


5'- GCAGCGTCAGATGTGTATAAGAGACAG--------------->
  |--5'- GTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGGTTTCAAACCCCGACCAGTTGGTAGGGGTTCCCATGTACTCTGCGTTGATACCACTGCTT -3'
                                                                                    <---------------CCTTAAGAGCCCACGGTTCC -5'

(5) Amplified cDNA bcgRNA products:

(i) cDNA amplification TSO product (173bp)


 5'- GCAGCGTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGGTTTCAAACCCCGACCAGTTGGTAGGGGTTCCCATGTACTCTGCGTTGATACCACTGCTT -3'
 3'- CGTCGCAGTCTACACATATTCTCTGTC[16-bp cell barcode][12-bp UMI]AACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCCCAAAGTTTGGGGCTGGTCAACCATCCCCAAGGGTACATGAGACGCAACTATGGTGACGAA -5'

(ii) cDNA amplification additive primer product (113bp) (Main product)


 5'- GCAGCGTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGG -3'
 3'- CGTCGCAGTCTACACATATTCTCTGTC[16-bp cell barcode][12-bp UMI]AACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCC -5'

(6) Perform a Feature PCR using Feature SI Primers 2 (PN-2000099) and RPI-index (i7) primer:

(i)


 5'- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG--------------->
                                    5'- GCAGCGTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGGTTTCAAACCCCGACCAGTTGGTAGGGGTTCCCATGTACTCTGCGTTGATACCACTGCTT -3'
                                    3'- CGTCGCAGTCTACACATATTCTCTGTC[16-bp cell barcode][12-bp UMI]AACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCCCAAAGTTTGGGGCTGGTCAACCATCCCCAAGGGTACATGAGACGCAACTATGGTGACGAA -5'
                                                                                                                       <---------------ACCTTAAGAGCCCACGGTTCC
                                                                                                                                                            TTGAGGTCAGTG[i7]TAGAGCATACGGCAGAAGACGAAC -5’

(ii)


 5'- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG--------------->
                                    5'- GCAGCGTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGG -3'
                                    3'- CGTCGCAGTCTACACATATTCTCTGTC[16-bp cell barcode][12-bp UMI]AACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCC -5'
                                                                                                                       <---------------ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTG[i7]TAGAGCATACGGCAGAAGACGAAC -5’

(7) (optional) P5+P7 PCR:


 5'- AATGATACGGCGACCACCGA--------------->
 5'- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[16-bp cell barcode][12-bp UMI]TTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
 3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGCAGCCGTCGCAGTCTACACATATTCTCTGTC[16-bp cell barcode][12-bp UMI]AACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTG[i7]TAGAGCATACGGCAGAAGACGAAC -5’
                                                                                                                                                             <---------------AGAGCATACGGCAGAAGACGAAC -5’

(8) Final library structure (~192bp):


 5'- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
 3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGCAGCCGTCGCAGTCTACACATATTCTCTGTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNAACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5’


        Illumina P5      Illumina Nextera Read1    Cell barcode   UMI         10x CS1   Feature Barcode   Illumina smRNA Read2   i7      Illumina P7

bcgRNA library Sequencing

(All primers are part of the Illumina sequencing kit.)

(1) Add Read 1 primers to sequence the first read (sequence 16-bp cell barcode and 12-bp UMI, 28 cycles):


                                   TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG---------------------------->
  3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGCAGCCGTCGCAGTCTACACATATTCTCTGTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNAACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5’

(2) Add Sample Index sequencing primer to sequence the 8-bp sample index (8 cycles):


                                                                                                                                     TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC------->
  3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGCAGCCGTCGCAGTCTACACATATTCTCTGTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNAACGATCCTGGCCGGAATTTCGNNNNNNNNNNNNNNNACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5’

(3) Cluster regeneration, add Truseq Read 2 primer to sequence the feature barcode read (min 15 cycles):


  5'- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTGCTAGGACCGGCCTTAAAGCNNNNNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                                      <--------------ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTG