drop_seq/drop-seq.human_text.txt

Drop-seq

Protocol

Drop-seq profiles single-cell RNA expression by co-encapsulating single cells and barcoded primer beads in nanoliter droplets. One aqueous flow contains cells, and the other contains barcoded primer beads suspended in lysis buffer. After droplet formation, cells lyse inside droplets and released mRNAs hybridize to bead-bound barcoded oligo-dT primers. Droplets are then broken, beads are collected and washed, and mRNAs attached to beads are reverse transcribed in bulk to form STAMPs, single-cell transcriptomes attached to microparticles. After exonuclease I treatment, bead aliquots are PCR amplified. The amplified cDNA is then prepared for sequencing using Nextera XT tagmentation with custom primers.

The bead-bound primers contain a common PCR handle, a cell barcode, a UMI, and an oligo-dT capture region. The cell barcode identifies the bead/cell of origin, and the UMI identifies individual captured mRNA molecules. Drop-seq libraries are sequenced paired-end. Read 1 is 20 bp: bases 1–12 are the cell barcode and bases 13–20 are the UMI. Read 2 sequences the cDNA insert and is used to identify the gene of origin.

Key oligo and library-related sequences

The explicit oligo sequences are provided in Table S6 of the supplemental information.

1. Barcoded Bead SeqA

Source sequence:

5'-Bead-Linker-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGTJJJJJJJJJJJJNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'

2. Barcoded Bead SeqB

Source sequence:

5'-Bead-Linker-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACJJJJJJJJJJJJNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'

3. Template_Switch_Oligo

Source sequence:

AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG

4. TSO_PCR

Source sequence:

AAGCAGTGGTATCAACGCAGAGT

5. P5-TSO_Hybrid

Source sequence:

AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT*A*C

6. Nextera_N701

Source sequence:

CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG

7. Nextera_N702

Source sequence:

CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG

8. Nextera_N703

Source sequence:

CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG

9. Read1CustomSeqA

Source sequence:

GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGT

10. Read1CustomSeqB

Source sequence:

GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC

11. P7-TSO_Hybrid

Source sequence:

CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGT*A*C

12. TruSeq_F

Source sequence:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T

13. CustSynRNASeq

Source sequence:

CGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGTAC

14. UMI_SMARTdT

Source sequence:

AAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTT

Step-by-step library generation

Step 1. Barcoded bead synthesis

Input substrate:

* Toyopearl beads / functionalized microparticles

Added oligos/reagents:

* reverse-direction phosphoramidite synthesis reagents

Molecular event:
Barcoded primers are synthesized directly on beads. Each bead receives many copies of the same cell barcode through split-and-pool synthesis. After barcode synthesis, each primer receives an 8-base UMI and an oligo-dT capture region.

Product structure:

Bead linker

* PCR handle
* cell barcode
* UMI
* oligo-dT capture sequence

Step 2. Droplet generation and mRNA capture

Input substrate:

* single-cell suspension
* barcoded primer beads

Added oligos/reagents:

* Drop-seq lysis buffer
* droplet generation oil

Molecular event:
Cells and barcoded beads are co-encapsulated in droplets. Cells lyse inside droplets, and polyadenylated mRNAs hybridize to the oligo-dT region of the bead-bound primers.

Product structure:

bead-bound primer

* captured polyadenylated mRNA

Step 3. Droplet breakage and reverse transcription

Input substrate:

* mRNA-bound beads from droplets

Added oligos/reagents:

* Template_Switch_Oligo
* reverse-transcription reagents

Molecular event:
Droplets are broken and beads are collected. Reverse transcription is performed on bead-bound mRNAs. Template switching introduces a template-switch sequence downstream of the synthesized cDNA.

Product structure:

PCR handle

* cell barcode
* UMI
* oligo-dT
* cDNA insert
* template-switch-derived sequence

Step 4. Exonuclease I treatment and PCR amplification

Input substrate:

* reverse-transcribed STAMPs

Added oligos/reagents:

* Exonuclease I
* TSO_PCR primer
* KAPA HiFi HotStart ReadyMix

Molecular event:
Exonuclease I removes unused primers. Bead aliquots are PCR amplified using the template-switch PCR primer.

Product structure:

amplified Drop-seq cDNA

Step 5. Nextera XT tagmentation and library PCR

Input substrate:

* amplified Drop-seq cDNA

Added oligos/reagents:

* Nextera XT reagents
* P5_TSO_Hybrid
* Nextera_N701 or other Nextera index primer

Molecular event:
3' cDNA fragments are prepared for sequencing using Nextera XT tagmentation. Custom primers replace the kit oligos to enrich Drop-seq cDNA fragments and add Illumina sequencing adapters and sample index sequence.

Product structure:

P5 side

* Drop-seq read 1 priming region
* cell barcode
* UMI
* oligo-dT / cDNA insert
* Nextera read 2 side
* sample index
* P7 side

Step 6. Sequencing

Input substrate:

* final Drop-seq sequencing library

Added oligos/reagents:

* Read1CustomSeqA or Read1CustomSeqB, depending on bead sequence version

Molecular event:
The library is sequenced paired-end. Read 1 is primed with a custom Drop-seq Read 1 primer and reads the cell barcode and UMI. Read 2 reads cDNA sequence for transcript/gene identification.

Read structure:

Read 1: 20 bp total

* bases 1–12: cell barcode
* bases 13–20: UMI

Read 2:

* cDNA insert sequence
* 50 bp in the human-mouse experiment
* 60 bp in the retina experiment

Final canonical library structure

Simplified segment-level structure:

P5 + Drop-seq Read 1 priming region + cell barcode + UMI + oligo-dT / cDNA insert + Nextera Read 2 side + i7 sample index + P7

Sequencing read interpretation:

* Read 1: 12-bp cell barcode + 8-bp UMI
* Read 2: cDNA insert used for gene identification
* i7 index read: sample index from Nextera index primer

Human-curation notes

1. The Drop-seq paper describes the bead primer architecture as a PCR handle, cell barcode, UMI, and oligo-dT capture sequence.
2. The supplemental protocol explicitly provides the oligo sequences in Table S6.
3. Barcoded Bead SeqA and Barcoded Bead SeqB differ in the sequence immediately upstream of the cell barcode. SeqA is used with Read1CustomSeqA, and SeqB is used with Read1CustomSeqB.
4. Table S6 shows both Barcoded Bead SeqA and SeqB with a 12-base cell barcode region followed by an 8-base UMI region. If an OCR output shows fewer J characters for SeqB, that should be treated as a text extraction issue.
5. The source states that Read 1 is 20 bp, with bases 1–12 corresponding to the cell barcode and bases 13–20 corresponding to the UMI.
6. The protocol uses Nextera XT library preparation with custom primers P5_TSO_Hybrid and Nextera_N701 in place of the standard kit oligos for the main library construction.
7. Nextera_N702 and Nextera_N703 are printed in Table S6 and used in specific multiplexed/contamination experiments, but Nextera_N701 is the main primer described for standard Drop-seq library construction.
8. Modified bases such as rG in Template_Switch_Oligo and * in P5_TSO_Hybrid, P7_TSO_Hybrid, and TruSeq_F should be preserved as printed.